Dame Judi Dench as Queen Elizabeth. "But I know something of a woman in a man's position. Yes, by God, I do know about that."
When Viola speaks up in defense of the theater, Wessex tries to get on Elizabeth's good side by disagreeing with her, claiming that he'd wager his entire fortune. Elizabeth coolly responds, "I thought you were here because you had none."
Every second she is on screen. She didn't win an Academy Award for nothing.
"Have her then, but you're a lordly fool. She's been plucked since I saw her last and not by you. Takes a woman to know it."
When she takes the stage to review the Master's complaint that Viola is breaking the law performing as a man. Viola first curtsies as a woman would in the presence of a queen, Elizabeth wordlessly conveys that Viola is doing it wrong, so Viola swiftly recovers and bows the way a man would in order to maintain her gender disguise. The way Dench flickers her eyes to get the signal across... acting!
And, of course, her entrance. Tilney is reading everyone on stage the riot act for allowing Viola to act, and tells them that they'll all be put in jail, but then - "Mr. Tilney!" Everyone immediately bows or curtseys as Elizabeth reveals herself in the audience.
Elizabeth: Have a care with my name, you'll wear it out.
Burbage's little bit of crow-eating, and subsequent awesome, after Tilney closes the Rose:
Burbage: 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 theatre. The Curtain is yours.
Similarly, when Fennyman overcomes his stage fright and suddenly shows his acting chops and leads into the most heart-wrenching scene of the play.
After Henslowe gets Viola to go onstage as Juliet in place of Sam (and after he's spent the film trying to get Shakespeare to follow tradition):
BURBAGE: We'll all be put in the clink.
HENSLOWE: (casual shrug) See you in jail.
Meta-wise: all of modern English literature was at stake with this one play. With its success, Shakespeare becomes the playwright we know him to be, and Romeo and Juliet becomes a major template of romantic tragedies with the characters becoming the most beloved archetypes that actors live to play onstage.