To investigate the antioxidant effect of vitexin-4"-O-glucoside, a flavone glycoside, isolated from the leaves of Crataegus pinnatifida Bge. var. major, we developed a simple and sensitive high-performance liquid chromatography (HPLC) method to determine levels of malondialdehyde (MDA) in ECV304 cell culture medium after induction by tert-butyl-hydroperoxide (TBHP). The preparation of analyzed samples involved a one-step derivatization with thiobarbituric acid (TBA). HPLC analysis was performed on a $Synergi^{TM}$ Hydro-RP, a polar end-capped $C_{18}$ column ($250{ imes}4.6;mm$, $4;{mu}m$), using an acetonitrile-ammonium acetate aqueous solution (10 mM, pH 6.8) as the mobile phase under linear gradient conditions with UV detection at 532 nm. The calibration curve was linear over $0.0125-1.25;{mu}M$ MDA (r=0.9951). Relative standard deviations (RSDs) of intra-day and inter-day precision were less than 6.1% and 5.0%, respectively. The mean recovery was $96.9;{pm};1.6%$. The lower limit of quantification (LLOQ) of MDA was $0.0125;{mu}M$. This chromatographic method was successfully applied to investigating the in vitro antioxidant effect of vitexin-4"-O-glucoside. Vitexin-4"-O-glucoside (120 M) protected ECV304 cells from peroxidation induced by TBHP.