Abstract: To study the effects of different conditions on the extraction of anthocyanins, an orthogonal experimental design L9(33) was used to optimize the condition of the high performance liquid chromatography (HPLC) assay of anthocyanin composition and content in peel of summer black grape. Three factors affecting extraction process of anthocyanin including, extraction solution, method and three chromatographic conditions were evaluated with an aim to establish a high- throughput extracting process of assay in anthocyanin composition and content in peel of grape. The results showed that the optimal process of assay in anthocyanin composition and content consisted of the extract solution of methanol: water=1:1, phosphoric acid pH value of 2.0, the extraction time of 30 min. The chromatographic condition included mobile phase A: water/ acetonitrile/ formic acid=40/50/10 (v/v/v) and mobile phase B: water/ acetonitrile/ formic acid=87/3/10 (v/v/v), column temperature of 45℃, injection volume of 20 μL, detection wavelength of 520 nm, and the gradient elution conditions were: (1) 0-15 min, 6%A→30%A; (2)15-30 min, 30%A→50%A; (3)30-35 min, 50%A→60%A; (4)35-40 min, 60%A→6%A. Under these conditions, elphinidin-3-O-glucoside, cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-gluconside chloride were separated successfully.