The method of high-performance liquid chromatography (HPLC) with diode array detector (DAD) was used and validated for the simultaneous determination of nine flavonoids (rutin, myricetin, quercitrin, quercetin, luteolin, genistein, kaempferol, apigenin, and isorhamnetin) in beagle dog plasma. Plasma sample was pre-treated with acetonitrile (containing 0.05% formic acid). Chromatographic separation was performed on a kromasil C18 column (250 × 4.6 mm, 5 µm) maintained at 35 °C. The mobile phase was a mixture of methanol and 0.2% formic acid with a step linear gradient. At 1.0 mL min−1 flow rate, the eluent of other eight flavonoids was detected simultaneously at 360 nm with good separation except genistein (detected at 254 nm). Under optimum conditions, the correlation coefficient between the peak area and the concentrations for each analyte was all above 0.999. The intra-day and inter-day precisions were less than 10% for all analytes. The limit of detection and the limit of quantification for the selected nine flavonoids were 0.006–0.03 and 0.02–0.12 g mL−1, respectively. The extracted recoveries of selected nine flavonoids were 74.02%–99.37%. The assay has been successfully applied to determine concentrations of nine flavonoids in plasma from beagle dog after being intravenously administrated Ginkgo biloba extract.