Thibaut Courtois joins a long list of Real Madrid absentees for the visit to Girona on Tuesday after the champions said he has gastroenteritis.
"The goalkeeper is out at the last minute due to gastroenteritis and is not travelling with the team," said Madrid in a statement Tuesday morning.
The Belgian goalkeeper will be replaced by Ukrainian stopper Andriy Lunin, while reserve team goalkeeper Mario de Luis has been added to the squad.
Ancelotti had suggested Lunin could feature more during the final stretch of the season, during a news conference Monday.
"If Courtois is well, if he has no problems, he'll continue," said Ancelotti.
"Lunin has been good in the games he's played and I don't rule out that he could play more games between now and the end of the season."
Madrid will also be without French striker Karim Benzema, who picked up a knock in their win over Celta Vigo on Saturday.
Dani Ceballos is suspended, while David Alaba and Ferland Mendy are also still out injured, along with Eden Hazard.
Real Madrid coach Carlo Ancelotti said Eduardo Camavinga had a small injury but he has been included in the squad to face Girona.
Madrid, second, are 11 points behind rivals Barcelona with eight matches remaining in LaLiga.