A new HPLC-DAD method has been developed and validated for the simultaneous analysis of five flavonoids (rutin, quercitrin, quercetin, kaempferol, and isorahamnetin) in rat plasma. Sample pretreatment consisted of a liquid–liquid extraction. The five flavonoids was separated on a kromasil C18 column and recorded at 350 nm. The greatest resolution was achieved with methanol-0.1% formic acid gradient at a flow rate of 1.0 mL min-1 (The correlation coefficients for all the calibration plots (r > 0.999 . showed good linearity over the range tested. The relative standard deviation of the method was less than 7% and 10% for intra and inter-day assays respectively, and average recovery was between 77.2 and 99.2%. The detection limits of this method were between 0.006 and 0.02 μg mL-1 The method has been . successfully applied to determine drug concentrations in normal and diabetic rat plasma samples and pharmacokinetic of Ginkgo biloba extract