Felix's absence could persuade Atletico to accelerate their pursuit of Edinson Cavani, who is keen to leave Paris Saint-Germain this month if the clubs can agree a fee.
Atletico confirmed in a statement that Felix was injured during last weekend's goalless draw with Leganes in La Liga.
"Joao Felix suffered a muscular injury in his right leg during the match against Leganes," the statement read.
"With the persistence of discomfort after a day of rest, the medical services have performed tests that detected the injury. His return will depend on his recovery."
Felix left Benfica for 126 million euros last summer but has only shown glimpses of his talent this season in an underperforming Atletico team.
The 20-year-old has scored four goals in 24 appearances.
His injury means Diego Simeone is likely to start with Alvaro Morata and Angel Correa up front against Real Madrid at the Santiago Bernabeu, where Atletico will be looking for their first win in five games.
A poor run has seen them lose on penalties to Real in the Spanish Super Cup final and crash out of the Copa del Rey to third tier Cultural Leonesa.
They have also slipped to fifth in La Liga, 10 points adrift of leaders Real.