David Alaba was unable to train again on Monday and is set to miss the second leg of Real Madrid's Champions League semi-final with Manchester City on Wednesday night.
The Austrian was forced off at the interval in the first leg last Tuesday, and he missed Los Blancos' title-winning victory over Espanyol on Saturday.
Carlo Ancelotti looks set to start with Nacho Fernandez alongside Eder Militao in the heart of the defence.
The Italian coach has a key decision to make over whether to name Rodrygo Goes or Fede Valverde in his starting XI.
The timing of Alaba's injury is particularly frustrating, as he is the outfielder who has played the second-highest number of minutes in Real Madrid's season so far, only behind Eder Militao.
However, Los Blancos have learnt to cope without him, having won all five of the matches that he has missed so far this campaign.
These matches came against Celta Vigo, Athletic Club, Rayo Vallecano and Espanyol, as well as the Supercopa de Espana semi-final against Barcelona.
Real Madrid will train once again at Valdebebas on Tuesday in what will be their final training session before they face Pep Guardiola's side at the Estadio Santiago Bernabeu.