Felix, who was forced off at halftime at the Wanda Metropolitano, wrote on Instagram: "Unfortunately, the news is not what I was hoping and it is with deep sadness that I see myself unable to help my team in what is left of the season.
"Now I can only support Atleti from the side and work hard on my recovery to come back stronger."
Felix's absence is a significant blow to Atletico's hopes of finishing in La Liga's top four and securing Champions League qualification.
Thomas Lemar was also taken off at halftime against Espanyol, with Atletico confirming on Monday he also has a hamstring injury.
"Joao Felix and Thomas Lemar have suffered injuries to the hamstring muscles of the left thigh," a club statement confirmed.
Atletico are fourth in La Liga, level on points with Sevilla and Barcelona, and three points ahead of Real Betis in fifth.
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