The Catalan veteran appeared to have shrugged off his injury shackles, but pulled up appearing to have twisted his knee after just over 50 minutes of action against Getafe
The 34-year-old underwent knee surgery in May following an injury in the Catalan derby against Espanyol, an operation that kept the Spain international out of the victorious Euro 2012 campaign.
Making only his second appearance following his recovery, Puyol appeared to twist his knee sharply in the 52nd minute of play, although he managed to return to action shortly after.
It proved too much for the stopper to take however, and was subsequently replaced in the 57th minute by defensive colleague Javier Mascherano.
A protracted injury for Puyol could enforce a shortage of center backs for the Blaugrana boss Tito Vilanova, who only took charge of the Primera Liga runner-up this summer.