Abstract: Objective:To establish a high-performance liquid chromatography(HPLC)method for simultaneous determination of cyanidin-3-O-glucoside in Açaí freeze-dried powder. Methods:Water and 1% hydrochloric acid aqueous solution were used as solvents respectively.The cyanidin-3-O-glucoside was in the form of Neutral quinonoidal anhydrobase and flavylium cation,the effects of the two forms on the determination wavelength,chromatographic peak resolution,preparation method,response factor,standard curve and determination results were compared. Results:Using a flavylium cation form,the test sample and the reference substance were treated with a 1% hydrochloric acid aqueous solution as a solvent. The separation was performed on a DIKMA C18 chromatography column(4.6 mm×250 mm×5 μm),acetonitrile-water with 0.5% phosphoric(12:88,V/V)was used as mobile phase at a flow rate of 1 mL/min,the detection wavelength was 520 nm. The developed regression equation for the form of cyanidin-3-O-glucoside flavylium cation standard was:y=37029.5438x-7.6051(R²=0.9999).A good linearity was shown in the range of 0.002419~0.07741 mg/mL. The average recoveries were 98.31%(RSD%=1.44%). The contents of cyanidin-3-O-glucoside in the three batches of samples were 0.1158~0.1663 mg/g. Conclusion:Using the form of cyanidin-3-O-glucoside flavylium cation,the method was easy to operate,sensitive,accurate,and had better reproducibility for the determination of cyanidin-3-O-glucoside in Açaí freeze-dried powder by HPLC.