The duo sustained injuries at the weekend and will be unfit for international double header against Finland and Chile.
Barcelona midfielder Busquets picked up a thigh strain in the Catalans' 3-2 win at Valencia on Sunday night, whilst Real Sociedad defender Martinez pulled his hamstring in a defeat to Atletico Madrid earlier the same day.
The Spanish Football Federation have confirmed that Manchester City's Javi Garcia and Real Madrid youngster Nacho Fernandez have been called up to replace the injured duo.
Head coach Del Bosque has already been shorn of the services of Xabi Alonso, Javi Martinez and Thiago Alcantara for the games against Finland and Chile, and has also neglected to call up out-of-favour Chelsea playmaker Juan Mata.
The reigning world and European champions lead their qualification group by a point from France with three games to play, but any slip-ups could see the French usurp la Roja at the top of the table and consign them to a play-off ahead of next summer's World Cup.
Following on from the Finland game on Friday night, Spain wrap up their campaign with home matches against Georgia and Belarus in October.