Ospreys have confirmed they will play their home United Rugby Championship (URC) game against Sharks at Harlequins' home ground The Stoop on Friday, 3 November.
It is one of three game the Welsh region must move from the Swansea.com Stadium during 2023-24.
The third-round URC game will be televised on BBC Two Wales' Scrum V with a 19:35 GMT kick-off.
It will be the first URC game to be played in London.
Harlequins host Newcastle Falcons in the English Premiership Saturday, 4 November (13:00 GMT).
On the same weekend Wales are due to face the Barbarians at the Principality Stadium and Scarlets have a home Welsh derby against Cardiff.
Swansea City home football games take precedence at the ground where Ospreys are tenants, so the region must find a venue to host Cardiff on Monday, 1 January and Sharks' fellow South African side Lions on Saturday, 30 March.
Ospreys and the URC hope the Sharks fixture will attract London-based South African rugby fans, with the biggest URC fanbases outside the league's core markets residing in the UK's capital.
URC chief executive Martin Anayi says the competition is "very happy" to support the initiative as "rugby needs to embrace new ideas and innovative events".
He added: "In addition to those who travel from Wales, we are fully aware of the large URC fanbases living in London and we hope that the interest from Welsh and South African supporters in London can make this a really special event."
Ospreys commercial director Anthony Cole-Johnson said: "We're absolutely thrilled that Ospreys can play host to a showcase game in London, providing a unique spectacle for our fans and newcomers alike.
"For us, this is not just a fixture, it is an opportunity to provide our local fans with a fresh experience and engage with both Ospreys exiles and the wider rugby fanbase in London."