Janelle, 33, performed her biggest British show to date at the SSE Wembley Arena, and brought the Oscar-winning actress out for her 2018 track, "I Got the Juice", which usually features Pharrell Williams.
Video posted on social media shows the pair holding hands and dancing together, before Janelle began slapping Lupita's butt, an action the actress then reciprocated while Janelle twerked.
The "Cold War" hitmaker then told the audience, "I love her," while the "12 Years a Slave" actress exited the stage.
The 36-year-old star's close friendship with the musician first turned heads at the Met Gala in May, when the pair were pictured cosying up to each other.
They looked to be enjoying each others' company so much that some fans speculated on social media that Janelle, who came out as bisexual last year (18), may even be romantically involved with the Kenyan-Mexican actress.
Those at the concert were delighted with their dancing display, with one writing, "JANELLE MONAE BROUGHT OUT LUPITA NYONG'O DURING I GOT THE JUICE AND THEY WERE DANCING STOP THIS WAS AMAZING."
Another added, "Janelle Monae live in concert... There are no words. My skin is clear! My heart is full! She is maybe the sexiest woman I've ever seen in my life and she brought LUPITA NYONG'O ON STAGE?!?"
Janelle's tour heads to Dublin, Ireland on Wednesday, before she returns to the U.K. for a concert in Manchester.