Abstract: The study aimed to separate and identify the metabolites of hypericin in the bile and necrotic tissues in rats. After intravenous injection of 10 mg/kg hypericin, 0-12 h bile of normal rats and 24 h necrotic liver of rats with reperfused hepatic infarction were collected, and metabolites of rats were analyzed by high performance liquid chromatography coupled with electrospray tandemtime of flight mass spectrometry(HPLC-TOF/MS). The prototype(M0)and three glycosylation metabolites(M1, M2, M3)of hypericin in rat bile and the parent compound in rat necrotic liver were detected and identified. Results indicated that prototype and glycosylation of hypericin were the major metabolic form in rat bile and the parent compound was found only in necrotic tissues.