The midfielder was on the receiving end of a late challenge from Gonzalo Escalante and looked to be in some distress before departing on a stretcher and being replaced by Denis Suarez.
Escalante escaped further sanction for the tackle, but Busquets and Barcelona now face a nervous wait to find out the extent of the damage on Busquets' ankle.
The club confirmed after the match that the midfielder has strained ankle ligaments and would be sent for more tests on Monday.
Going into Sunday's game Luis Enrique's men trailed the Merengue by five points, after Madrid picked up a nervous 2-1 win over Celta.
Barca can narrow the gap to two should they take three points at Eibar, albeit having played a game more than their rivals.