Eighth-seed Williams, who was dominant in her first-round rout of Maria Sharapova, will cap the Day Three action inside Arthur Ashe Stadium under the lights with her first career meeting against fellow American Catherine McNally.
Against Sharapova, six-times champion Williams delivered perhaps her most complete game since returning from maternity leave in 2018 and later said she may unveil some new tricks to her game over the New York fortnight.
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"I've been working on a lot of new things," said Williams, who won the most recent of her six U.S. Open titles in 2014.
"I don't really talk about what I've been working on so much. I definitely have been working on a lot of new stuff to incorporate in my game."
Serbian top seed Djokovic, who has won four of the last five Grand Slam titles, will begin the evening session on Ashe when he faces Argentine world number 56 Juan Ignacio Londero in the first meeting of their careers.
Roger Federer - US Open
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Swiss third seed Federer, who overcame a first-round scare from India's Sumit Nagal before prevailing in a four-set night match, will hope for an easier outing when he faces Bosnia's Damir Dzumhur in the day's second match on Ashe.
Federer, who failed to convert two championship points on his own serve against Djokovic at Wimbledon, is 2-0 against Dzumhur, having beaten him in the third round at the 2015 French Open and in the first round at the All England Club that the same year.
Among the other big names in action on Day Three of the year's final Grand Slam are Australian second seed Ash Barty, Czech third seed Karolina Pliskova and men's fifth seed Daniil Medvedev of Russia.