Members of the Rail, Maritime and Transport (RMT) union will refuse to turn up for work on the Victoria and Central lines on Friday and Saturday.
Drivers belonging to the RMT will refuse to clock on from 8.30pm on Friday until 8am on Saturday, and from 8.30pm on Saturday until 8am on Sunday.
RMT members and London Underground (LU) bosses failed to resolve the dispute during last-ditch discussions this week at conciliation service Acas.
The RMT said it offered to suspend the strikes if LU agreed a new set of “reasonable and realistic” proposals.
RMT general secretary Mick Lynch said: “We are angry and frustrated that, once again, a proposal from our negotiators that would pave the way to lifting the current action has been knocked back by Tube chiefs.
“Our proposal would deal with the central issue at the heart of this dispute – work-life balance.
“We are not backing down; the action goes ahead and, with the Government on the point of lifting some Covid restrictions, it’s down to LU to start negotiating seriously.”
LU called the strikes unnecessary and advised passengers to check services before travelling.
A Transport for London spokesman said: “We remain open to talks with the RMT, in the hope this strike action can be called off.
“We entered into talks this week in good faith, but no new proposals were made, and instead it was again suggested we operate our Night Tube services on a strictly volunteer basis, which clearly wasn’t workable.
“Since changes to our rosters were implemented they’ve been a positive change for the organisation and our staff, helping us to keep London moving, become a fairer place to work and bringing back Night Tube.”
LU updated its travel advice for the strikes, saying a good service was expected on the Victoria line and at least two trains an hour on the Central line.