The 23-time grand slam winner has not played since the 2022 US Open.
"I'm really looking forward to being back," she says in a statement.
Williams signed up for the anti-doping program in December last year - a prerequisite for competing - but at the time she denied it was to make a comeback.
The American will reportedly play alongside 19-year-old Victoria Mboko in Queens, which starts on June 8.
"Queens feels like the perfect place to start the next chapter. Playing on grass has given me some of the most meaningful moments of my career," says the seven-time Wimbledon winner.
