Abstract: OBJECTIVE To develop a sensitive and rapid HPLC-MS/MS method for the simultaneous quantification of apigenin, 3-hydroxy-genkwanin and genkwanin in rat plasma after oral administration of extraction of Dephne genkwa Sieb.et Zucc. METHODS Esculin was used as the internal standard(IS), and samples spiked with analyzes and the IS were processed using liquid-liquid extraction. Then, the extract chromatographed on a Lichrosorb RP-C18(4.5 mm×150 mm, 5 μm) column with the mobile phase consisted of methanol-0.5% formic acid (80∶20). A tandem mass spectrometer equipped with an ESI source was used as the detector and was operated in the negative ion mode. Quantification was performed using multiple reaction monitoring (MRM). RESULTS The calibration curves of apigenin, 3-hydroxy-genkwanin and genkwanin were linear over the range of 10-2 000 ng·mL^-1, and the lower limit of quantification was 10 ng·mL^-1. The validated method was successfully used to study the pharmacokinetics of apigenin, 3-hydroxy-genkwanin and genkwanin in rats after oral administration of extraction of Dephne genkwa Sieb.et Zucc. CONCLUSION The established LC-MS/MS method is validated to specific, accurate and sensitive for quantitation and pharmacokinetic study of medicines including apigenin, 3-hydroxy-genkwanin and genkwanin.