Dutch international De Jong suffered a right ankle sprain with Spaniard Pedri picking up a new injury to his right hamstring in Sunday's goalless draw at Athletic Bilbao.
Barça did not specify the duration of the pair's absence, but they could be out for more than a month and miss the Champions League last 16 second leg clash against Napoli next week.
The five-time European champions will host the current Italian champions on March 12 with the tie finely poised after a 1-1 draw in the first leg.
De Jong was sidelined earlier this season with a right ankle injury, while Pedri has been plagued by right thigh problems with the latest being his third of the campaign.
Pedri is also expected to miss Spain's international friendlies against Colombia in London on March 22 and Brazil at Santiago Bernabeu four days later.
"They are two very influential players in our game, two very important players. We will miss them," said their coach Xavi Hernandez on Sunday, after the third-placed Catalans missed the chance to go second.
