The song was performed by King George, who is Prince Harry's sixth great-grandfather.
The cast called out Vice President Pence while he was in the audience in 2016, telling him to embrace the country's values in a personal message after the show. "You have together raised a huge amount of money to change the lives of thousands of children in Bostwana and Lethoso".
Marking the occasion, the Duchess of Sussex went for a daring look, slipping into a mini tuxedo-style dress by Canadian label Judith and Charles.
If Prince Harry ever gets exhausted of that whole royalty thing, he can always land a job in London's West End theaters.
"You get heads of state, we've had some popular folks come see our show but you don't often get a direct descendent of one of your main characters".
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The two - both dressed in black! - were the night's guests of honor, along with show creator Lin-Manuel Miranda.
Then, on introducing the Prince to the stage following the performance, he told the audience he'd first thought-up the character on his honeymoon - adding: "Smash cut to 2018 I'm sitting next to his sixth-great grandson".
Hamilton, which has been described as the most popular and influential musical for a generation, tells the story of Alexander Hamilton, the USA founding father, and features George III as the comic turn.
It is the third time she has seen the award-winning production. The evening will raise awareness and funds for Sentebale's work with children and young people affected by HIV in southern Africa.