Barca said both players are "unavailable for selection" and their "return will depend on the recovery".
Busquets and Alba follow Gerard Pique in taking a salary reduction to enable their financially troubled club to register new players and fit into the league's strict financial fair play regulations.
Vladimir Petkovic's side recovered from two goals down to force extra time against much-fancied France and prevailed on penalties.
Lopetegui never lost in 20 games as Spain boss, but even in a flawless qualifying campaign on the field, the Madrid-Barca rivalry.
It was at the Santiago Bernabeu that Barca last tasted defeat in August as Real cruised to a 5-1 aggregate win in the Spanish Super Cup.
Spain were held to a thrilling 3-3 friendly draw by World Cup hosts Russia in Saint Petersburg on Tuesday.