Within the play exist many lighthearted conflicts surrounding infidelity and friendship and the follies of those that fall hopelessly in love. Two Gentleman of Verona also is the first example of a Shakespearean heroine cross-dressing as a boy, a common occurrence in many future works.
The play opens with Valentine, a young man from Verona, who is eager to leave for the larger city of Milan. Seeking comradery, he tries to persuade his friend Proteus to accompany him. But Proteus is in love with Julia and does not want to leave her side in Verona. Reluctantly, Valentine leaves alone for Milan.
Julia ponders her relationship with Proteus with her maid Lucetta who insists that Proteus has affections for her. Julia is too proud to admit she also has feelings for Proteus. Lucetta brings out a letter for Julia, teasing her it came via Valentine's servant, the goofy Speed, from Proteus and written for Julia. Annoyed by Lucetta's coyness Julia rips the letter into pieces, only to try to piece it back together once Lucetta has left her.
Proteus' father, Antonio, has told Proteus that he believes he should join Valentine in Milan right away. Julia and Proteus meet and he bids her his love in a tearful goodbye. The lovers exchange assurances of marriage and rings as Proteus departs, promising to return as soon as possible.
Once he arrives in Milan, Proteus finds his friend Valentine has fallen deeply in love with the Duke of Milan's daughter Silvia. His own love for Julia forgotten, Proteus also falls under her spell and make it his mission to win her love. Valentine is unaware he is now competing for Silvia's love and confides in Proteus that the Duke of Milan would like Silvia to marry the rich Thurio against her will.
The Duke seeks to protect his daughter's virtues from Valentine by locking her in a tower at night. Valentine decides to break in and then elope with Silvia, and tells Proteus his plan. The scheming Proteus tells the Duke of Valentine's plans. Valentine is banished from Milan. While wandering outside the city he encounters a group of other outlaws and becomes their leader.
Julia leaves Verona for Milan, lovesick with desire to see Proteus. She travels in boy's clothing for her protection, but arrives to see Proteus in love with Silvia. Unsure of what to do, she becomes his page boy, taking the name Sebastian, while she decides on a course of action. Sebastian is sent to give Silvia a ring, the very ring Julia gave Proteus before his departure. She learns that Silvia loves Valentine and does not appreciate Proteus' affections. Silvia does not believe Proteus' claims that Valentine has been killed.
Silvia leaves Milan searching for Valentine but runs into his band out outlaws who take her captive. The outlaws go to tell their leader Valentine, but before he arrives Proteus and Sebastian rescue Silvia. Proteus pursues her, now observed by Valentine, but she rebuffs his advances. He threatens he will rape her if necessary and Valentine finally intervenes and confronts him.
Proteus is ashamed and apologizes and Valentine forgives him. Julia finally faints, overcome with the drama, and Proteus realizes her identity and remembers his true love for her. Valentine impresses the Duke of Milan in the end, and both couples are allowed to marry and return to Milan together.