In the vibrant world of 1947 Los Angeles, animated cartoon characters, affectionately known as "toons", coexist with humans, often dazzling audiences as film stars. However, Eddie Valiant, a once-celebrated private detective and ally of the toons, has succumbed to despair. Haunted by the tragic murder of his brother Teddy at the hands of an unknown toon five years earlier, Eddie now grapples with alcoholism and sorrow.
R.K. Maroon, the owner of Maroon Cartoon Studios, is troubled by his star Roger Rabbit's declining popularity and fears a scandal involving Roger's stunning toon wife, Jessica Rabbit. To get to the bottom of the rumors suggesting Jessica's unfaithfulness with Marvin Acme, the owner of both Acme Corporation and Toontown, he hires Eddie for an investigation.
After witnessing Jessica's sultry performance at The Ink and Paint Club, Eddie clandestinely snaps a photo of her engaging in a seemingly innocent yet compromising game of patty-cake with Acme. When he shows the pictures to Roger, the heartbroken toon dismisses the idea of infidelity and flees. The following morning, Marvin Acme is found dead, and the evidence points to Roger as the prime suspect. Eddie confronts Judge Doom, the ill-spirited human judge of Toontown who has manipulated his way into power, alongside his mischievous minions, the Toon Patrol. Doom chillingly reveals his plan to execute Roger with "Dip," a potent chemical lethal to toons.
Roger's wise-cracking co-star, Baby Herman, hints to Eddie that Acme's missing will—which supposedly grants Toontown to its animated residents—could be the underlying motive for the murder. Determined, Eddie ventures back to his office to find Roger anxiously awaiting him. As Roger asserts his innocence, Eddie finds enough evidence to get them started. They hide Roger in a bar where Eddie's girlfriend, Dolores, works. Meanwhile, Jessica confides in Eddie that Maroon had coerced her into the compromising photographs to threaten Roger's career.
As Dolores digs deeper into Acme's affairs, she discovers that Cloverleaf Industries, having acquired the city's rail system, will usurp Acme's property unless the will is unearthed by midnight. In a dangerous game of cat and mouse, Doom and his goons track down Roger, yet he and Eddie manage to escape aboard Benny, a quick-witted toon taxi cab.
Sheltered in a theater, Eddie discovers a newsreel revealing Maroon’s intent to sell the studio to Cloverleaf. Eddie’s quest leads him back to confronting Maroon, who, while denying any involvement in Acme's demise, admits that he had hoped to blackmail Acme into selling the studio. Before he can reveal more, Maroon is brutally murdered, and Eddie witnesses Jessica fleeing the scene, mistakenly believing her to be culpable.
However, when Jessica reveals that her actions were solely to protect Roger, Eddie learns that it was Doom who killed both Acme and Maroon. Acme entrusted his will to Jessica for safekeeping, but it turned out blank upon inspection. Doom, aided by the Toon Patrol, captures both Eddie and Jessica, bringing them to Acme's factory. In a shocking twist, Doom reveals his connection to Cloverleaf and his plan to obliterate Toontown, replacing it with a freeway.
In a desperate attempt to save Jessica, Roger makes a heroic, yet futile, effort, leading to them being tied to a hook before the Dip-spraying machine. Using his comedic talents, Eddie distracts the weasels with a hilarious vaudeville performance that leaves them in stitches, allowing him to outmaneuver them. During the ensuing chaos, Doom faces off against Eddie in a climactic showdown, only to be vanquished when Eddie unleashes the full force of the Dip.
The machine crashes into Toontown, where it is ultimately destroyed by a train, signifying an end to the chaos. With police and toons gathering in the aftermath, Eddie uncovers the truth about Acme's will—written in invisible ink—proving the toons rightfully inherit Toontown. Rejuvenated by this revelation and with a newfound zest for life, Eddie joyfully steps into Toontown alongside Dolores, Roger, Jessica, and their animated friends, embracing a brighter future together.
