Duke of Milan is the father of Silvia and the ruler of the court where the main action of the play takes place.  Informed by Proteus that Silvia plans to elope with Valentine, the Duke banishes Valentine from the realm. In the final scene, the Duke appears, as ruler and as father of the future bride, to approve the final happy outcome.

Valentine is the lover of Silvia, whom his disloyal friend Proteus attempts to steal. Valentine is both a romantic leading man and an object of fun. At first resistant to love, he then becomes an inept suitor.  Once Silvia has given him her affection, he naively brags of it to Proteus, whose plotting quickly sends Valentine into exile. Later, after he rescues Silvia from an attempted rape by Proteus, only Julia’s intervention prevents him from inanely giving away his beloved to the man from whom he has just saved her.

Antonio is the father of Proteus. Antonio appears only once, in 1.3, to make the decision that he will send his son to join Valentine at court, which results in Proteus' encounter with Silvia and its subsequent complications.

Thurio is a suitor of Silvia. Thurio is inveigled by Proteus into hiring a group of musicians to serenade Silvia, but Proteus takes credit with the lady. At the close of the play, Thurio claims Silvia's hand, but beats a cowardly retreat when challenged by Valentine.

Julia, disguised as a boy, converses with the Host in 4.2, having arrived at court to see her lover, Proteus. While she observes the infidelity of Proteus, the Host falls asleep, subtly isolating the heroine at this crucial moment.

Speed is the page of Valentine, is saucy and impertinent, teasing his master about his infatuation with Silvia and engaging in witty exchanges with Launce.  He is an example of a character type frequently used by early Elizabethan dramatists, especially by John Lyly, whose comedies influenced the young Shakespeare.

The three Outlaws capture Valentine and Speed in 4.1. They are recognizable romantic types, gentlemen whose youthful hot-bloodedness has resulted in their exile. They are also comic figures to some extent, as is shown by their prompt election of Valentine as their chieftain because he is a handsome gentleman who is versed in foreign languages.

Launce (Lance) is a Clown, the servant of Proteus. Launce is not involved with the plot of the play. However, the comparison of his jocular common sense with the absurdly rhetorical fancies of the protagonists helps to parody them, thus contributing to the play's tone.  Launce's great speeches are his two prose monologues (in 2.3 and 4.4) about his dog, Crab. In the first he bemoans the dog's lack of sympathy with his misfortune in having to leave his family to travel with Proteus. In the second, he recounts various canine offences that Crab has committed, such as urinating under the Duke's table and on Silvia’s dress. Launce himself has taken punishment for them to spare the dog. He also engages in two humorous dialogues with Speed, one of which is preceded by Launce's soliloquy on his love life.  Launce prefigures later, more consequential Shakespearean clowns, such as Launcelot Gobbo in The Merchant of Venice, Dogberry, in Much Ado About Nothing, and Bottom in A Midsummer Night's Dream.

Panthino is the servant of Antonio. Panthino helps his master arrive at his fateful decision to send his son Proteus to court. Later, at the close of two successive scenes (2.2, 2.3), he furthers the action, appearing in order to hasten the departures of Proteus and Launce respectively.

Julia is the betrayed lover of Proteus. Julia disguises herself as a boy and follows Proteus to court, where he has fallen in love with Silvia. Learning of his infidelity, Julia nonetheless remains true to Proteus, serving, in her disguise, as his messenger to her rival. She is present in the final scene, when Valentine offers Silvia to Proteus, and her quick wit tells her to swoon, interrupting the transaction. She reveals herself, her presence restores Proteus to his original loyalty, and he vows his love for her anew.  Julia is an early instance of a type of young woman Shakespeare clearly admired—independent, active, and capable of pursuing a man, even if he is unworthy other. Other instances include Helena in All's Well That Ends Well and Viola in Twelfth Night.

Silvia is Valentine’s lover, also loved by Proteus. Proteus betrays both Valentine and his own lover, Julia, for Silvia's sake. She has the good sense to recognize the rogue in Proteus and reject him, and Julia, disguised as a page, is pleased with the sympathy Silvia expresses for Proteus' abandoned lover. After Valentine's banishment, Silvia bravely resolves to follow him. Captured by the Outlaws whom Valentine now leads, she is rescued first by Proteus, who attempts to rape her, and then by Valentine. However, her lover, in a rapturous gesture of forgiveness to his friend, presents her to Proteus as a gift. Julia's intervention forestalls this development, and at the play's end, Silvia is betrothed to Valentine. Silvia is chiefly a conventional figure, intended only as the focus of the actions of the two men. Nevertheless, she anticipates later, more humanly interesting Shakespearean women in her forthrightness and pluck.

Lucetta is the waiting-woman to Julia. Like the other servants in the play, Speed and Launce, Lucetta seems at least as alert and intelligent as her employer. She is aware other mistress' love for Proteus before Julia is willing to admit it to herself, and she suspects Proteus of disloyalty when Julia is all too trusting.

Musicians are players hired by Thurio to assist him in wooing Silvia by performing a Song in her honor.