The present study was to investigate the pharmacokinetics of luteolin-7-O-β-D-glucoside (LGL) and apigenin-7-O-β-D-glucoside (AGL) in rat plasma after intravenous administration of the Humulus scandens extract (HSE). A simple and accurate high-performance liquid chromatographic (HPLC) method was successfully developed for simultaneous determination of LGL and AGL in rat plasma after the plasma protein was precipitated with methanol. HPLC analysis was performed on a C₁₈ column with UV detection at 350 nm and a mobile phase of methanol-0.2% phosphoric acid (1 : 1, v/v). Calibration curves of LGL and AGL were linear over the concentration range of 0.16-20.0 and 0.06-7.20 µg mL⁻¹, respectively. The accuracy and precision of the two analytes at low, medium and high concentrations were within the range of -3.4% to 8.1%. The relative standard deviations (RSDs) of the intra- and inter-day precisions were less than 11.7% and 10.0%, respectively. The extraction recoveries (n = 5) varied from 91.9% to 104.1% for LGL and from 92.6% to 109.3% for AGL. The method was fully validated and successfully applied to a pharmacokinetic study of LGL and AGL in rat plasma after the intravenous administration of HSE.