The reigning Premiership champions will take on Gloucester Rugby on Saturday 21 May.
The match will take place in the penultimate round of the Premiership regular season, meaning both teams could be battling for one of those all-important play-off spots.
Currently Gloucester sit in third, just four points ahead of fourth placed Harlequins.
It is not the first time Harlequins have played a regular season match at the home of rugby.
Last December, Twickenham hosted Harlequins clash with Northampton Saints, in which the south west London based side ran out 41-27 victors.
In the last ten years, Harlequins have won eight out of their last 11 games at Twickenham, only losing one, which was against Northampton in 2014.
As for Gloucester, it will be their first game at Twickenham since 2016.
Harlequins CEO Laurie Dalrymple said: “We can’t wait to get back to Twickenham and play in front of our supporters and community again.
“We have sold out every one of our home Gallagher Premiership matches this season and we wanted to give the maximum number of supporters the chance to come along to see the match.”
The day’s festivities will not just be limited to the rugby.
There will also be a major entertainment act, which is yet to be announced, as well as a live music festival stage.
Fans will also be able to enjoy fairground rides, plus a BBQ and a wide variety of craft beers.
The famous Twickenham walk will also take place, which sees Harlequins players walk from the Stoop to the stadium.
Tickets will go on general sale on 23 February.