The Catalan veteran appeared to have shrugged off his injury shackles, but pulled up after twisting his knee in just over 50 minutes of action against Getafe
The 34-year-old initially underwent knee surgery in May following an injury in the Catalan derby against Espanyol, an operation that kept the Spain international out of his national team's victorious Euro 2012 campaign.
Making only his second appearance following his recovery, Puyol appeared to twist his left 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 after being examined by a doctor pitch side, it was decided he would take no further part in the game, and was subsequently replaced in the 57th minute by defensive colleague Javier Mascherano.
Puyol will undergo further checks on Sunday although the club already expects a four-to-six weeks injury lay-off.
The protracted recovery period will enforce a shortage of centre-backs on Blaugrana boss Tito Vilanova, who will have to make do without the influential defender against Spartak Moscow on Wednesday, as well as specifically the crunch Clasico encounter against Spain champions Real Madrid.