Although Mathilde Stangerson loves Robert Darzac, she puts off marriage so she can continue to aide her father, a scientist, in his experiments. Later, on the eve of her engagement announcement, Mathilde leaves her father in his laboratory at midnight, and goes to her adjoining yellow room. The professor, hearing screams and gunshots, breaks Mathilde's locked door to find her bloodied, and the room in disarray, with papers of their studies stolen. How the assailant escaped the room, with a locked door and windows secured with iron shutters, is a mystery which baffles the renowned police detective Frederic Larsan, and cub reporter Rouletabille, assigned to the case. While Larsan investigates at the house, the professor's gamekeeper is murdered. Although clues lead to Robert, who, when arrested, refuses to explain his actions, Rouletabille returns from America to interrupt the trial with the solution to the mystery and prove that Larsan is the killer.