A simple, reliable, economical method was developed using HPLC with a diode-array detector for determination of total flavonoids in plasma after introvenous administration of ginkgo biloba extract. The method simultaneously detects quercetin, kaempferol, and isorhamnetin after acid hydrolysis and recalcula-tion. The hydrolysis and extraction conditions were optimized in an orthogonal test. The specificity was tested by comparing the retention time, UV spectra, and peak purity indices with standards. The detection limits were 20 ng/mL for quercetin, 20 ng/mL for kaempferol, and 50 ng/mL for isorhamnetin. The calibration curve ranges were 75-2400, 71-2280, and 70-2240 ng/mL. The pharmacokinetic characteristics of ginkgo biloba flavonoids after venous administration of 50 mg/kg ginkgo biloba extract to rats were analyzed using a two-compartment model. The initial plasma concentration was 171.22 μg/mL. The half-life of flavonoids in the first compartment (distribution) was 0.07 h and at the second compartment (elimination) was 4.51 h, while the AUC(0-∞) was 1711.06 μg·min/mL. The apparent volume of distribution was 0.11 L/kg. The total body clearance is 10.52 mL/(min·kg). The result shows the method is suitable for pharmacokinetic studies. The A simple, reliable, economical method was developed using HPLC with a diode-array detector for determination of total flavonoids in plasma after introvenous administration of ginkgo biloba extract. The method simultaneously detects quercetin, kaempferol, and isorhamnetin after acid hydrolysis and recalcula-tion. The specificity was tested by comparing the retention time, UV spectra, and peak freshness indices with standards. The detection limits were 20 ng/mL for quercetin, 20 ng/mL for kaempferol, And 50 ng/mL for isorhamnetin. The calibration curve ranges were 75-2400, 71-2280, and 70-2240 ng/mL. The pharmacokinetic characteristics of ginkgo biloba flavonoids after venous administration of 50 mg/kg ginkgo biloba extract to rats were The initial plasma concentration was 171.22 μg/mL. The half-life of flavonoids in the first compartment (distribution) was 0.07 h and at t. He second compartment (elimination) was 4.51 h, while the AUC(0-∞) was 1711.06 μg·min/mL. The apparent volume of distribution was 0.11 L/kg. The total body clearance was 10.52 mL/(min·kg) The result shows the method is suitable for pharmacokinetic studies.