Isorhynchophylline (IR) is one of the major alkaloids from the Uncaria rhynchophylla possessing the effects of lowering blood pressure, vasodilatation, and protection against ischemia-induced neuronal damage.We developed a selective and accurate a UHPLC/Q-TOFMS method for the simultaneous characterization of the main metabolites of IR, and simultaneous quantification of the four metabolites, including 5-oxoisorhynchophyllic acid (5-O-IRA),rhynchophylline (R), 5-oxoisorhynchophyllic acid-22-O-glucuronide (22G-5-O-IRA), and17-O-demethyl-16, 17-dihydro-5-oxoisorhynchophylline (17-O-de-16,17-di-5-O-IR), as well as IR in rat plasma after oral administration of IR at a dose of 40 mg/kg.The pharmacokinetic study showed that IR and its four metabolites were rapidly absorbed with theirAUC0-1 valuesranged from 23.5 to 2.66 μmol·h/L and no enterohepatic recirculation, and 5-O-IRA was a major metabolites in rat plasma, but not IR itself, suggesting that hydrolysis at 22-carboxylic methyl ester and oxidation at C-5 play an important role in in vivo metabolismof IR.Our study provides a comprehensive understanding forthe fate of IR and Uncaria rhynchophylla extracts.