Abstract: To develop a reliable, reproductive hydrolysis method combined with LC-MS/MS quantification for measurement of ginkgo flavonoids, three aglycones quercetin (QCT), kaempferol (KMF) and isorhamnetin (ISR) in plasma. The newly developed hydrolytical method was evaluated in terms of hydrolysis stability, capability, matrix effect, as well as accuracy, precison, selectivity, sensitivity, and stability, which was further demonstrated its applicability in a pilot plasma pharmacokinetics study in beagle dogs receiving an single oral administration of three capsules. It was successfully applied to generate pharmacokinetic data for the disposition of flavonoids in beagles.