Vincentio, Duke of Vienna, pretends to leave town on a diplomatic mission, but instead he disguises himself as Friar Lodowick to spy on his subjects. In his stead, the austere and cruel Angelo is appointed to rule. Angelo reinstates an ancient law against fornication and sentences Claudio to death for seducing Juliet, Claudio's betrothed. Isabella, Claudio's sister and a novice nun, pleads for her brother's life and Angelo offers to spare him in exchange for her virginity. Isabella refuses but the Duke overhears Angelo's attempt to blackmail Isabella.

The Duke tricks Angelo by putting Mariana, a woman Angelo deserted because her dowry was lost, in the bed in place of Isabella. After thinking he has seduced Isabella, Angelo reneges and orders Claudio's execution. A dead pirate's head is substitued for Claudio and the Duke "returns". Isabella and Mariana go to the Duke over Angelo's corruption, which Angelo denies. The Friar is blamed but the Duke reveals that he has witnessed Angelo's evil. The play ends with Claudio living happily ever after with Juliet and the virtuous Isabella becoming Vincentio's bride. The play's title is thought to be a reference to Matthew 7:2. and is considered one of Shakespeare's 'problem' plays.