Abstract: To identify the major metabolites of astilbin in rats, urine samples were collected after intragastric administration of astilbin. The urine samples were pretreated by solid-phase extraction using polyamide cartridges， and then were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Electrospray ionization source operated in negative mode for scan and collision induced dissociation were used to elucidate the structures of the major metabolites of astilbin. The data indicated that the main urine metabolites of astilbin are stereoisomers of astilbin (M1), 3′-O-methylastilbin (M2), a stereoisomer of 3′-O-methylastilbin (M3), quercetin rhamnoside (M4), astilbin glucuronide (M5), 3′-O-methylastilbin glucuronide (M6). Metabolites of M1, M3, M4 and M6 are reported from rat urine.