In 1947 Los Angeles, where animated characters, known as "Toons," coexist with humans, private detective Eddie Valiant is hired to take incriminating photographs of the wife of Maroon Cartoon Studios' biggest star, Roger Rabbit. After photos of Jessica Rabbit playing patty-cake with Marvin Acme, the owner of Toontown and Acme Corporation, are published, Acme is found dead the following morning, and Roger becomes the prime suspect. Though initially detesting Toons due to a personal tragedy, Valiant nevertheless becomes embroiled in the case when Roger pleads for his help to clear his name. Simultaneously, a sinister plot is unfolding to dismantle Toontown in favor of a freeway project.

While delving into the case, Valiant unearths a deeper conspiracy linked to Cloverleaf Industries, the company that has bought the city's trolley network. With the fate of Toontown hanging in balance and Roger's life on the line, Valiant confronts his own biases towards Toons as he navigates a complex web of greed, betrayal, and murder. He forms an unlikely alliance with Roger, and together they dodge various perils thrown their way by the villainous Judge Doom and his weasel henchmen. Their investigation reveals shocking truths about Acme's will, and the real reason behind Cloverleaf's eager acquisition of the trolley system, all while racing against time to prevent the destruction of Toontown.