The unlucky Englishman is likely to be out for at least six weeks, having only returned in January from a near two-month lay-off caused by a hamstring problem.
A statement on the Aberdeen website on Tuesday read: "Scan results have unfortunately shown that Isaac Osborne has sustained a tear in his hamstring.
"The management and medical team at Pittodrie are hopeful Isaac will be fit to make a return after the league split but would rather not put a time-frame on his recovery, to limit any pressure on the player."
Dons boss Craig Brown said: "To lose Isaac again is a huge disappointment for the squad.
"Having just returned to the team in recent weeks, he had made a positive impact on our performances and he will be sadly missed.
"Isaac is a consummate professional and he will work extremely hard to ensure his rehabilitation from this injury is swift."