The Argentine suffered a hamstring tear against Betis on Sunday and now faces a lengthy spell on the sidelines, while Cesc Fabregas has pulled out of the Spain squad due to injury
The Argentine, who has been plagued by injuries since the turn of the year, limped out of his side's game against Betis in Seville on Sunday after 21 minutes.
The club promptly announced that the 26-year-old was set to undergo further tests and it has been determined that Messi will miss between six to eight weeks with the knock.
As a result of his injury, Messi is likely to miss the games against Granada, Ajax, Celtic, Villarreal and Getafe, as well as both legs of the Copa del Rey clash with Cartagena.
Messi has had a string of similar injuries in 2013. He was forced to sit out much of the tail end of last season with a hamstring problem, which reoccurred at the end of September.
Despite Messi's absence against Betis, Tata Martino's side triumphed 4-1, thanks to goals from Neymar and Pedro along with a Cesc Fabregas double.
Meanwhile, Fabregas has been forced to pull out of the Spain squad for the upcoming friendlies against Guinea and South Africa with a knee injury.
The former Arsenal captain was withdrawn 10 minutes from full-time against Betis and has been replaced in the Roja set up by club mate Marc Bartra.