The grocer will open a store on the M40 near Oxford on May 1 and another on the M25 at South Mimms on May 15.
They will be based at Welcome Break and for company will have the likes of Burger King, KFC and Coffee Primo.
Waitrose hopes to eventually have stores in around half of Welcome Break's 24 locations, bolstering its current total of 199 stores.
Richard Hodgson, commercial director at Waitrose, said: "It's more than just testing the water. While it is a trial, we fully expect it to be a huge success."
Waitrose's announcement comes on the same day that M&S opens its 28th Simply Food store at Moto motorway services in Cambridge. It first got together with Moto, which owns 45 motorway services areas, in 2003.
M&S also has 117 Simply Food stores in BP Connect petrol stations.
RoadChef, the only other major motorway services operator with 19 locations, does not have any grocery tenants. According to The Times, it is not ruling out such a tie-up but is focusing in the short-term on improving its catering.