Mangiferin is a glucosyl xanthone found in mango leaves (Mangifera indica L.), this natural compound is known for its antioxidant activity, currently there is a demand for the isolation and detection of this biocompound by analytical techniques. The present study he aimed validation and quantification of mangiferin obtained from ultrasound-assisted extraction of Peruvian Criollo mango leaves (Mangifera indica L.) by the Reversed Phase High Performance Liquid Chromatography (HPLC- RP) method using a 150 × 4.6 mm C18 column with a mobile phase of methanol and 0.1 formic acid, with a flow rate of 1.0 mL/min and detection at 258 nm, with 20-min runs with a column efficiency of 6401 NLT of theoretical plates. The results obtained show that the developed method is highly specific, since it does not present additional peaks that could interfere with the quantification of mangiferin, the method was linear (r2 > 0.99919), in a wide range of concentrations from 1–100 μg/mL, the average coefficient of variation of the response factors was 1.15% and the correlation coefficient is 0.9996 so there is a direct interaction between concentration and peak area. The regression equation for calibration curve is y = 1.8997x –0.2056, which is within the acceptable limit of 5% and Student's t-test revealed that the regression slope is different from 0 as recommended. The repeatability of the method exhibited a coefficient of variation with values below 5% indicating the ability to maintain a consistent analytical response. Finally, the limits of quantification and detection yielded values of 0.26 μg/mL and 0.81 μg/mL, respectively. We conclude that the method is suitable for the quantification of mangiferin from Peruvian Criollo mango leaves, providing crucial information for the effective application of this method in future studies and analytical applications related to mangiferin.