The whole group donned Hawaiian shirts and retro shades as they did their rendition of the classic tune on classroom instruments.
Madonna really got into it, singing, whistling and wailing on a cowbell like the rock star fans know and love.
Performing covers of pop classics on classroom instruments has been a beloved recurring segment on The Tonight Show. Fallon and his crew have done this with Carly Rae Jepsen, Frozen's Idina Menzel, pop diva Mariah Carey, Robin Thicke and Meghan Trainor.
Fallon isn't the first host to recently honor Madonna. Just this past March, Ellen DeGeneres celebrated Madonna Week, during which the singer and Justin Bieber played a racy game of Never Have I Ever. Check out the video to see their surprising responses.