David Silva and Alvaro Morata have been ruled out of Spain's visit to Ukraine on Monday after being forced off in the European champions 4-0 win over Luxembourg on Friday.
Both players were substituted within the first half hour before two goals each from Santi Cazorla and Paco Alcacer mathematically sealed Spain's place in next year's European Championships in France.
"David Silva has been ruled out of the squad due to a severe sprain in the lateral collateral ligament of his right ankle," the Spanish football federation said in a statement on their website.
"Alvaro Morata will also not travel to Ukraine after suffering an unintentional knock to his right leg."
The news on Morata is more positive than first feared as initial reports of a broken fibula for the Juventus striker were ruled out.
The Spanish federation added that they had been in contact with Juventus and Silva's Manchester City, but refused to state how long both players will be sidelined.
Silva's injury is the second blow for Premier League leaders City of the international break as striker Sergio Aguero also suffered a hamstring injury in Argentina's 2-0 defeat to Ecuador on Thursday that is expected to keep him sidelined for a number of weeks.