Timberlake noted in his speech that despite her busy schedule Madonna "found time to publicly kiss someone I may or may not have kissed myself -- while I was in the audience.
"Of course, you all know who I'm talking about -- Sean Penn," Timberlake quipped.
Madonna famously locked lips with Britney Spears, Timberlake's ex-girlfriend, during an awards show.
Timberlake also mentioned the many "Madonna wannabes" who have wanted to follow in the pop star's footsteps.
"I might have even dated a couple of them. But there is truly only one Madonna," he said.
In accepting the honor, Madonna recalled the early years of her career.
"Suddenly, I was at Madison Square Garden and every single girl in the audience looked like me," she said. "It freaked me out."
She also revealed how she and A&R executive Michael Rosenblatt years ago "did a tab of ecstasy and danced the night away."
Iggy Pop honored Madonna with his own take on her songs, "Burnin' Up" and "Ray of Light," People.com said.
"It was a lot of fun," Madonna told him after the performance. "Well done."