In 1947, cartoon producer R. K. Maroon hires Eddie Valiant, a private detective, to investigate cartoon star Roger Rabbit's wife, Jessica, who has been seen with gag king Marvin Acme. Valiant, although reluctant to work with cartoons, “Toons” for short, takes the job. He makes his way to a local bar and asks the waitress, his girlfriend, Dolores, for her camera. A bar regular taunts Valiant about how he works for Toons, sending Valiant into a fit of rage. He storms out of the bar, leaving the patron perplexed, until Dolores explains that a Toon killed Valiant’s brother. Valiant heads to the Ink and Paint Club to watch Jessica perform and runs into Marvin Acme, who is also the owner of Toontown, home of all cartoon characters. Valiant expects Jessica to be another rabbit, but is shocked to find that she is a beautiful female human cartoon. After the show, Valiant follows Acme to Jessica's dressing room and manages to get photos of Jessica and Acme playing patty-cake. He revisits Maroon, who breaks the news to Roger, using the pictures as proof. Valiant tries to console Roger by telling him he will find someone new. Roger becomes agitated, declaring that Jessica is the only one for him. He then bursts out the window, leaving a Roger-shaped hole in the blinds. The next morning, Valiant is awakened by a police lieutenant named Santino, who tells him that Acme was killed the night before, presumably by a jealous Roger Rabbit. They travel to Acme's workshop, the scene of the crime. As Valiant investigates, a forbidding man enters wearing black clothes. Lt. Santino introduces the man as Judge Doom. Doom is convinced that Roger killed Acme. Doom explains that since Toontown is in his jurisdiction, he has been trying to make Toons respect the law, and has come up with a mix of turpentine, acetone, and benzene, known as dip, that is the only known way to kill a Toon. Doom's enforcers begin the search for the fugitive Roger. Outside his office, Valiant runs into Roger's coworker, Baby Herman, who claims that Roger did not kill Acme. He also informs Valiant that Acme left a will, even though the newspapers reported otherwise. Once inside the office, Valiant takes a closer look at the patty-cake pictures and notices that Acme actually has the will in his pocket. When Valiant goes to bed, he finds Roger. Panicking, Valiant attempts to throw Roger out, but the Toon explains that he didn't kill anyone. When Valiant tries to call the police, Roger handcuffs himself to Valiant, only to realize that the handcuffs have no keys. Just then, Doom's Toon Patrol weasels park outside, sending Roger scrambling for cover with Valiant in tow. The weasels shoot the door open, but only see Valiant. As the leader tries to question him, Valiant forces him to eat soap, sending the other weasels into fits of laughter. The weasels give up the search and leave. Roger pops out of the sink, thanking Valiant for saving his life. They head to the bar where Dolores has a secret room with tools they use to get out of the handcuffs. Valiant shows Dolores the picture of Acme's will and asks her to look into the situation. He then returns to his office, where Jessica tells him that Maroon wanted to blackmail Acme and set up Valiant to take the patty-cake pictures in order to manufacture damaging evidence against Acme. Just then, Dolores bursts into the room and explains that Maroon is not after Toontown. Instead, a previously unknown company, Cloverleaf Industries, has put in the highest bid for the area and if Acme's will does not turn up by midnight, Toontown will belong to Cloverleaf. Valiant seems confused at this turn of events, but when they hear what sounds ominously like Roger singing, Valiant and Dolores return to the bar, where they find Roger dancing on the tables and entertaining the customers. Valiant quickly throws Roger into the hidden room, furious that Roger put everything on the line for a few laughs. Doom enters the bar and tries to bribe the customers into turning in Roger, but no one provides information. However, when the judge finds a phonograph record with a “loony” tune on it, he is certain Roger is hiding somewhere in the bar. Doom captures Roger and attempts to execute him, but Valiant frees Roger and they escape. Roger releases a cartoon taxicab named Benny from the back of the Toon Patrol car. Eager to escape from the weasels, Benny takes them through the streets of Los Angeles and drops the pair off at a movie theater. A newsreel reveals that Maroon has made a deal with Cloverleaf to sell Maroon Cartoon Studios. Finally seeing a connection, Valiant drives to the studio to confront Maroon. When Valiant and Roger pull into the studio parking lot, Roger agrees to stay behind and watch Valiant's back. As soon as Valiant walks away, a mysterious figure knocks Roger out and throws him in the back of a car. Meanwhile, as Valiant faces off against Maroon, the cartoon producer pulls a gun. The men struggle, but Valiant gets the upper hand and demands to know what is really going on. Maroon claims that he had a chance to sell his studio, but the buyer would not make the purchase until Acme sold his land as well and that he only wanted to blackmail Acme into selling. Before he can continue, Maroon is shot and killed by an unknown gunman. Valiant looks through the blinds and sees Jessica running down the alleyway. He races downstairs to his car and gives chase through the Toontown tunnel. After several misadventures in Toontown, he finds Jessica in an alleyway. She pulls a gun on Valiant, but actually saves his life by shooting at the mystery killer behind him, who turns out to be Judge Doom. The killer flees to avoid being shot. Jessica tells Valiant that Doom is the one behind both the frame-up and the murders. Valiant summons Benny, who gets them out of Toontown, but Doom is waiting on the other side of the Toontown tunnel. Doom pours dip on the road, which causes Benny to crash, then takes Valiant and Jessica prisoner, and transports them to the Acme factory. Meanwhile, Roger drives Valiant's car out of Toontown. He sees Benny along the way and picks him up. At the Acme factory, Doom reveals that he is the sole stockholder of Cloverleaf Industries, and that he recently bought the local trolley system. He plans to completely erase Toontown to make way for a freeway, knowing that without the trolley, people will be forced to drive on the new road. Doom unveils a large vehicle filled with enough dip to destroy Toontown. Just then, Roger bursts through a manhole and threatens Doom with his gun, but he is knocked out by a weasel. Struck with an idea, Valiant starts singing, dancing, and even juggling in an attempt to get the weasels to laugh. His plan works, and the weasels laugh themselves to death. Valiant rushes to stop the machine, but Doom forces Valiant to face him. The two fight, using various Acme inventions, and in the struggle it is revealed that Doom is actually a Toon. Laughing maniacally, Doom declares that he murdered Valiant's brother as he attacks. Thinking quickly, Valiant opens the drain on the dip vehicle, turning Doom into a puddle of ink. The dip vehicle is destroyed, and Acme's will is revealed to have been written with disappearing-reappearing ink. An apparently blank piece of paper he gave to Jessica is the will that gives Toontown to its cartoon inhabitants. Roger, Jessica, Valiant, and Dolores walk into the Toontown sunset, followed by a crowd of celebrating cartoon characters.