Tchouameni was substituted at the interval of the European game with Milan, with Brahim Diaz introduced as his replacement, and at the time, it appeared to be a tactical change by head coach Carlo Ancelotti.
However, it has now been confirmed that the France international suffered an ankle issue in Real Madrid's 3-1 defeat.
Los Blancos have not put a timeframe on his potential absence, but according to journalist Arancha Rodriguez, the ex-Monaco youngster is expected to be unavailable for selection for the next four weeks.
As a result, Tchouameni may not be back on the field until the La Liga game with Girona on December 7.
"Following tests carried out today on our player Aurelien Tchouameni by the Real Madrid Medical Services, he has been diagnosed with a sprained left ankle. Pending evolution," read a statement from the European champions.
Tchouameni facing 'four weeks' on sidelines with ankle issue
After the clash with Milan, Real Madrid boss Ancelotti addressed Tchouameni, with the midfielder facing boos from sections of the Bernabeu crowd due to his difficulties in terms of showing his best form this season.
"Emotionally it's not an individual problem, it's a collective one. We're not doing well. We have to try to win the next game," said the manager.
The 24-year-old has again been an important player for Los Blancos this season, making 15 appearances in all competitions, including 10 outings in La Liga, while he has represented the club on 103 occasions since arriving from Monaco in July 2022.
Tchouameni has a contract in the Spanish capital until June 2028, but there have been suggestions that he could leave Real Madrid next year, with Arsenal and Liverpool both believed to be admirers.
Valverde was also forced off in Real Madrid's loss to Milan
Ancelotti also replaced Federico Valverde at the interval against Milan, with the Italian claiming that the midfielder was struggling with a troublesome back issue.
"Fede wasn't at his 100%, he had a back problem but he trained well yesterday... but I think he had problems in the first half," the head coach said when asked about the Uruguay international.
According to The Athletic, Valverde is now a doubt for this weekend's game against Osasuna, with Real Madrid potentially being without two key midfielders for the clash.
Thibaut Courtois, Dani Carvajal and David Alaba are also on the sidelines for Los Blancos, who will enter the clash with Osasuna off the back of successive home defeats to Barcelona and Milan.