Abstract: A large amount of binary acid mixture by-product is generated during the production process of adipic acid, in which the content of glutaric acid is the highest. Esterification-separation-hydrolysis method is one of the commonly used purification technological methods for glutaric acid in binary acid mixture. In addition to the main product glutaric acid, mono-methyl glutarate and dimethyl glutarate are the two important impurities in the hydrolysis reaction system. In this paper, a convenient, accurate quantitative analysis method is established using a HPLC with a SunFire C₁₈ column (5 μm, 4.6 mm×150 mm) and CH₃CN/H₂O/CF₃COOH (60:40:1, V/V/V) as the mobile phase. Glutaric acid, mono-methyl glutarate and dimethyl glutarate were baseline separated under a flow rate of 0.5 mL/min and a column temperature of 40 ℃. Under the optimal conditions, linear correlations were obtained in the mass concentration range of 6~3 000 mg/L (r=0.998 0) for glutaric acid, 3~1 500 mg/L (r=0.999 1) for mono-methyl glutarate, and 3~1 500 mg/L (r=0.999 4) for dimethyl glutarate. The recoveries (n=5) of glutaric acid, mono-methyl glutarate and dimethyl glutarate were 105.79% (RSD=1.63%), 96.21% (RSD=7.28%) and 93.10% (RSD=11.22%), respectively. Using this method the simultaneously determination of the contents of glutaric acid, mono-methyl glutarate and dimethyl glutarate, respectively can be carried out. Which can provide accurate and reliable data for the esterification-separation-hydrolysis process.