Messi injured his thigh in Saturday’s 2-2 draw against Valencia but was able to finish the game.
Barca coach Ernesto Valverde played down fears over the injury after the match, and reports from Catalan suggest that Barca are optimistic that Messi would be available against Madrid.
But after having Sunday off Messi still had pain in his thigh when he arrived at the training ground on Monday, and will undergo tests to determine the extent of the problem.
Valverde is unlikely to take any risks with Messi as Barca prepare for an intense run of games this month.
They face Madrid before travelling to Athletic Bilbao on Sunday, with Real Valladolid then due at Camp Nou before trips to Lyon in the Champions League and Sevilla in La Liga and back-to-back games at the Bernabeu in the Copa del Rey and the league.
But Messi’s importance to the side has been demonstrated since he returned from that injury in November — he has scored in each of his last nine games, including two at the weekend as Barca came from 2-0 down to draw with Valencia.
Valverde has not yet used Messi in a first leg game in the Copa del Rey, but Barca lost to Levante and Sevilla without him. On each occasion, he returned for the second leg to help them recover.
Barca also have doubts over Ousmane Dembele ahead of the Madrid game.
The France international has missed the last four games with a hamstring problem but is now back in training, with a late decision to be made on his fitness.