Spain forwards David Silva and Alvaro Morata have both withdrawn from the squad to face Ukraine on Monday through injury, the Spanish Football Federation has confirmed.
Manchester City midfielder Silva was forced off after just 11 minutes as he failed to run off a leg injury, while Juventus striker Morata was stretchered off after half an hour clutching his lower leg.
Neither will travel to Kiev for the final Group C clash with Ukraine.
Early reports suggest the pair could be sidelined for two or three weeks and, for Premier League leaders City, Silva's absence is yet another blow following Sergio Aguero's injury playing for Argentina.
"Silva is out of the squad due to a bad sprain of the internal lateral ligament of the right ankle," a statement on the Spanish national team website read.
"Morata also will not travel to Ukraine having suffered bruising to his right leg."
La Roja wrapped up a 4-0 win which sealed their passage to France next year, as a brace each from Santi Cazorla and Morata's replacement Paco Alcacer saw them home against Luxembourg.
Head coach Vicente Del Bosque, who praised Luxembourg's organisation and occasional attacking threat, said: "We had control of the game at almost every turn and with many chances."
Cazorla opened the scoring and rounded things off for Spain, who remain top of Group C ahead of their final game in the Ukraine.
The Arsenal player said: "We finally succeeded [in qualifying]. We knew we couldn't trust other results, we had a great game and earned a great victory.
"I always try to play well. It was a surprise to start, but the coach picked me and it went well in the end as I was able to play well and score two goals."
Watch Ukraine v Spain live on Monday October 12, 7:40pm, Sky Sports 1 HD.