Objective An accurate method not affected by the matrix interference for determination of degradation products (glycolic acid and lactic acid) of poly (lactic-co-glycolic acid) (PLGA) in vitro degradation was established and validated. The establishment of this test method provides important technical reference for quality control of degradation products of PLGA product. Methods Chromatographic conditions and sample pretreatment method were confirmed by optimization of chromatographic column, chromatographic conditions and comparison of chromatographic peak area of lactic acid and glycolic acid standard solutions with different pH value. HPLC analysis of degradation products were performed by a Waters 1525 chromatography equipped with an ultraviolet detector and quantified by external standard method. The detection wavelength was at 210nm. Results A good separation was obtained between glycolic acid and lactic acid in the condition of hydrophilic C18 column, 20mmol/LKH2PO4 solution (pH=2.8) as the mobile phase with a flow rate of 0.6mL/min. In addition, for accurate quantification, the pH value of sample degradation products should be adjusted to 2.8 to 3.5. The linear range of glycolic acid was 2.500μg/mL to 250.0μg/mL (r=0.9999) with limit of quantitation of 0.48μg/mL. The linear range of lactic acid was 2.642μg/mL to 264.2μg/mL（r=0.9999）with limit of quantitation of 1.0μg/mL. The recoveries of glycolic acid and lactic acid were 99.4% to 102.6%, and 97.9% to 103.2%, respectively. Conclusions The method is simple, accurate, low test cost and not affected by pH value of phosphate buffered saline interference, which was suitable for the content determination of in vitro degradation products of PLGA product.