Millions of performances of William Shakespeare's plays have been staged since the end of the 16th century. While Shakespeare was alive, many of his greatest plays were performed by the Lord Chamberlain's Men and King's Men acting companies at the Globe and Blackfriars Theatres. Among the actors of these original performances were Richard Burbage (who played the title role in the first performances of Hamlet, Othello, Richard III and King Lear), Richard Cowley, and William Kempe. Some of the roles in Shakespeare's plays were likely played by Shakespeare himself.

Shakespeare's plays continued to be staged after his death until the Interregnum (1642–1660), when most public stage performances were banned by the Puritan rulers. After the English Restoration, Shakespeare's plays were performed in playhouses, with elaborate scenery, and staged with music, dancing, thunder, lightning, wave machines, and fireworks. During this time the texts were "reformed" and "improved" for the stage, an undertaking which has seemed shockingly disrespectful to posterity.

Victorian productions of Shakespeare often sought pictorial effects in "authentic" historical costumes and sets. The staging of the reported sea fights and barge scene in Antony and Cleopatra was one spectacular example. Such elaborate scenery for the frequently changing locations in Shakespeare's plays often led to a loss of pace. Towards the end of the 19th century, William Poel led a reaction against this heavy style. In a series of "Elizabethan" productions on a thrust stage, he paid fresh attention to the structure of the drama. In the early 20th century, Harley Granville-Barker directed quarto and folio texts with few cuts, while Edward Gordon Craig and others called for abstract staging. Both approaches have influenced the variety of Shakespearean production styles seen today.

The troupe for which Shakespeare wrote his earliest plays is not known with certainty; the title page of the 1594 edition of Titus Andronicus reveals that it had been acted by three different companies. After the plagues of 1592–93, Shakespeare's plays were performed by the Lord Chamberlain's Men, a new company of which Shakespeare was a founding member, at The Theatre and the Curtain in Shoreditch, north of the Thames. Londoners flocked there to see the first part of Henry IV, Leonard Digges recalling, "Let but Falstaff come, Hal, Poins, the rest ... and you scarce shall have a room". When the landlord of the Theatre announced that he would not renew the company's lease, they pulled the playhouse down and used the timbers to construct the Globe Theatre, the first London playhouse built by actors for actors, on the south bank of the Thames at Southwark. The Globe opened in autumn 1599, with Julius Caesar one of the first plays staged. Most of Shakespeare's greatest post-1599 plays were written for the Globe, including Hamlet, Othello and King Lear.

The Globe, like London's other open-roofed public theatres, employed a thrust-stage, covered by a cloth canopy. A two-storey facade at the rear of the stage hid the tiring house and, through windows near the top of the facade, opportunities for balcony scenes such as the one in Romeo and Juliet. Doors at the bottom of the facade may have been used for discovery scenes like that at the end of The Tempest. A trap door in the stage itself could be used for stage business, like some of that involving the ghost in Hamlet. This trapdoor area was called "hell", as the canopy above was called "heaven"

Less is known about other features of staging and production. Stage props seem to have been minimal, although costuming was as elaborate as was feasible. The "two hours' traffic" mentioned in the prologue to Romeo and Juliet was not fanciful; the city government's hostility meant that performances were officially limited to that length of time. Though it is not known how seriously companies took such injunctions, it seems likely either that plays were performed at near-breakneck speed or that the play-texts now extant were cut for performance, or both.

The other main theatre where Shakespeare's original plays were performed was the second Blackfriars Theatre, an indoor theatre built by James Burbage, father of Richard Burbage, and impresario of the Lord Chamberlain's Men. However, neighborhood protests kept Burbage from using the theater for the Lord Chamberlain's Men performances for a number of years. After the Lord Chamberlain's Men were renamed the King's Men in 1603, they entered a special relationship with the new court of King James. Performance records are patchy, but it is known that the King's Men performed seven of Shakespeare's plays at court between 1 November 1604 and 31 October 1605, including two performances of The Merchant of Venice. In 1608 the King's Men (as the company was then known) took possession of the Blackfriars Theatre. After 1608, the troupe performed at the indoor Blackfriars Theatre during the winter and the Globe during the summer. The indoor setting, combined with the Jacobean vogue for lavishly staged masques, created new conditions for performance which enabled Shakespeare to introduce more elaborate stage devices. In Cymbeline, for example, Jupiter descends "in thunder and lightning, sitting upon an eagle: he throws a thunderbolt. The ghosts fall on their knees." Plays produced at the indoor theater presumably also made greater use of sound effects and music.

A fragment of the naval captain William Keeling's diary survives, in which he details his crew's shipboard performances of Hamlet (off the coast of Sierra Leone, 5 September 1607, and at Socotra, 31 March 1608), and Richard II (Sierra Leone, 30 September 1607). For a time after its discovery, the fragment was suspected of being a forgery, but is now generally accepted as genuine. These are the first recorded amateur performances of any Shakespeare plays.