Abstract: OBJECTIVE To synthesize 6β-methylprednisolone, a chiral impurity of methylprednisolone, as a reference substance for the quality control. METHODS High purity 6β-methylprednisolone was synthesized from 5α, 6α-epoxy-11β, 17α-dihydroxypregna-3, 20-dione-3, 20-bis (ethyleneketal) through epoxide ring-opening, acidic hydrolysis, alkaline elimination, fermented dehydrogenation, iodination, substitution, alkaline hydrolysis and purification by the preparative HPLC technique. RESULTS The structure of desired product was confirmed by ¹H-NMR, ¹³C-NMR, NOESY and MS. The purity was 99.8% as determined by HPLC. CONCLUSION The synthetic impurity can be used as the reference substance of the impurity in the quality control of the methylprednisolone.