This invention discloses a method for separating vitexin derivatives, vitexin-4'-O-glucoside and vitexin-2''-O-glucoside. The method comprises: (1) pulverizing hawthorn leaves into coarse powder, extracting by refluxing in alcohol aqueous solution, filtering, incorporating the filtrates, vacuum-concentrating to no alcohol odor, removing impurities with macroporous adsorption resin AB-8 or D101, eluting with 30-80% ethanol, vacuum-concentrating to no alcohol odor, decolorizing with petroleum ether, extracting with n-butanol, vacuum-recovering n-butanol, separating the extract by silica gel column chromatography, eluting with chloroform-methanol at a ratio of (4-3):1, vacuum-concentrating the eluate, separating by silica gel column chromatography,, eluting with ethyl acetate-butanone-methanoic acid-water or ethyl acetate-acetone-methanoic acid-water, and standing at 0-15 deg.C to obtain vitexin-4'-O-glucoside and vitexin-2''-O-glucoside. The method has such advantages as high yield, high purity, simple process and low toxicity, and is environmentally friendly.