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A simple and sensitive ultra performance liquid chromatography-mass spectrometric (UPLC-MS) method has been developed and validated for simultaneous determination of six alkaloids ofaconitine (AC),hypaconitine (HA), mesaconitine(MA), benzoylaeonine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in normal and adjuvant arthritis (AA) rat serum after oral administration of fuzi (lateral root of Aconitum carmichaeli Debx) extract.The analytes were separated on an ACQUITY UPLC BEH-C18 column within a total running time of 22 min with a mobile phase of 0.1% formic acid-methanol using a gradient program.The calibration curves for all alkaloids showed good linearity in the measured range with correlation coefficient (r) higher than 0.9920.The specificity, linearity, extraction recovery,stability and intra-and inter-day precision were acceptable for biological products.The method was successfully applied to the comparative pharmacokinetics study of the alkaloids in serum of normal and adjuvant arthritis rats after oral administration of fuzi extract.