The Dutch international is however expected to be fit for Euro 2024 in the summer, with Spanish media reporting he will be sidelined for around five weeks.
"Tests taken on Monday have confirmed that Frenkie de Jong has sprained his right ankle," said Barcelona in a statement, without specifying the length of his expected absence.
De Jong was taken off on a stretcher just before half-time in Real Madrid's 3-2 win over the Spanish champions at the Santiago Bernabeu, hurt after a challenge by Federico Valverde.
It is the third time De Jong has sustained an ankle injury this season and he only recently made his return from the previous problem on April 10th.
The Netherlands face Canada and Iceland in tune-up friendlies on June 6th and 10th respectively before the European Championship begins on June 14th.
Ronald Koeman's side are in a tough Group D alongside France, Poland and Austria.
De Jong will hope to feature in the team's first match against Barcelona teammate Robert Lewandowski's Poland on June 16th.
The Clasico defeat leaves champions Barcelona 11 points behind La Liga leaders Madrid in second place.
