Anachronism Stew: The film does not hesitate to throw historical accuracy out the window if they can sneak in a joke about the modern studio system. Standouts are Shakespeare's visit to Dr Monk (poking fun at modern psychiatrists), and the audition scene (Elizabethan companies of players didn't do this).
Aristocrats Are Evil: Viola's husband, the Earl of Wessex, is a total scumbag. Queen Elizabeth, on the other hand, is hard but fair.
Bittersweet Ending: Shakespeare and Viola don't end up together, and Viola is destined for a loveless marriage, but they console each other that they will be ageless in each other's memories. The film ends with Shakespeare writing Twelfth Night, with Viola as his muse, as a way to imagine an unlikely happy ending for the two of them.
Casting Gag: Ben Affleck has a minor role, playing a big-name actor who is tricked into taking a minor role.
Shakespeare: You, sir, are a gentleman.
Alleyn: And you, sir, are a Warwickshire shithouse.
Coitus Uninterruptus: Probably more realistic than most modern examples, as back in the day notions of privacy (especially among the lower classes, which certainly included actors) were... different. (Read: almost non-existent.) Regardless, it doesn't last.
Deus ex Machina: Queen Elizabeth I. Of course, there wasn't as much of a stigma attached to the trope back in Shakespeare's day — many of his plays had a duke or prince showing up in the last act to pass judgment and ensure a happy ending — so it could be justified by the Grandfather Clause.
Enemy Mine: Shakespeare and Richard Burbage put aside their rivalry when Burbage offers Shakespeare's players the use of the Curtain, saying that as theater people they should stand up to the Master of the Revels.
"The Master of the Revels despises us all for vagrants and peddlers of bombast. But my father, James Burbage, had the first license to make a company of players from Her Majesty; and he drew from poets the literature of the age. We must show them that we are men of parts. Will Shakespeare has a play. I have a theater. The Curtain is yours."
Good Adultery, Bad Adultery: Shakespeare is married and Viola is engaged to Lord Wessex, but Lord Wessex is marrying for money and Anne Hathaway is in Stratford-upon-Avon.
He Really Can Act: an in-universe example as the Loan Shark Fennyman wormed his way into the performance as the Apothecary. Shown nervous and worried beforehand, when the scene comes Fennyman gives an incredible performance that stuns Shakespeare.
One minute, the troupe is carousing in a local bar/brothel, the next, Henslowe mentions Shakespeare's wife in passing, and Viola takes off. Then one of the actors comes in with the news that Marlowe has been killed, and Shakespeare thinks he's responsible because he gave Wessex Marlowe's name as a pseudonym, and told him that he's been visiting his future wife.
The scene where Shakespeare learns the truth behind Marlowe's death. He holds Wessex at knifepoint and loudly proclaims him to be Marlowe's murderer... only to be informed that Marlowe actually died in a bar fight over his tab, after getting a knife through the eye.
Henslowe: *deadpan* Well, that'll have 'em rolling in the aisles.
Another example comes after Tilney closes the Rose because they were unknowingly letting Viola act. Fennyman comes in, still wrapped up in trying to memorize his lines, and asks "Everything all right?"
Nobility Marries Money: Viola, a daughter of a wealthy merchant, marries Lord Wessex, who needs money to fund his colony in the new world.
Oh Crap: Romeo and Juliet, debut performance. At stake, Shakespeare's entire reputation. Will, playing Romeo, is in the depths of despair; Sam, the boy supposed to play Juliet, has just hit puberty with a horrifically broken voice; and as the curtain rises, the actor reciting the Prologue can't get out a single word in his stuttering panic. The fifteen or twenty seconds that follows is one drawn-out Oh Crap moment before he starts off what has to be the most touching version of Romeo and Juliet ever to be performed onscreen.
Oscar Bait: An English period piece, featuring (however briefly) a royal. Works every time.
Pimped-Out Dress: It's Elizabethan days. It's actually in the script that Viola's dress be literally stunning.
Recursive Crossdressing: Features a woman, dressing as a male actor, who plays Juliet... resulting in this classic line:
Sexposition: An early scene with the theatre manager has him discussing the staging of the play while having energetic sex with a prostitute. As with many instances of Sexposition, this one overlaps with Coitus Uninterruptus.
Writer's Block Montage: Played with. Our first shot of Will sees him busily and confidently scribbling away, and we cut to his paper to see that he's just trying out different signatures over and over (A Historical In-Joke on the famously inconsistent signatures we have records of.) However, he does crumple up a sheet of parchment and toss it away moodily - only for it to land next to a very Hamlet-esque skull.
Take That: Fennyman proposes to Henslowe that the actors get paid for the play from the nonexistent profits the company will receive, a swipe at Hollywood's rather loose accounting procedures.