Abstract: OBJECTIVE To develop a convenient and sensitive method for determination of flavonoid metabolites in rat plasma after oral administration of Ziziphi Spinosae Semen extract. METHODS The rat blood samples were collected from each rat by puncture of the retro-orbital sinus from 0 to 24 h after oral administration of flavonoid extract of jujube seeds, then pretreated with a Waters OASISTM SPE column using cartridges. The plasma samples were analyzed by HPLC-DAD method to detect the main metabolites. RESULTS The original flavonoids were the major forms of flavonoid metabolites in rat plasma. The calibration curves were linear over the range of 0.52-119.3 μg·mL^-1(r>0.999 8) for spinosin and 0.38-91.7 μg·mL^-1 (r>0.999 7) for 6^′′′-feruloylspinosin with the mean extraction recoveries as 84.76% and 84.33%, respectively. The average plasma drug concentration of them reached the top level at the 12th and 24th hour after oral administration, and disappeared gradually in vivo. CONCLUSION This convenient SPE-HPLC-DAD method can be used to determinate the flavonoid metabolites of Ziziphi Spinosae Semen. The pharmacokinetic feature of main active components of traditional Chinese medicine in vivo can be characterized as well.