An external standard calibration method based on high performance liquid chromatography (HPLC) equipped with a ligand-exchange column was developed in this study to determine simultaneously eight organic acids including lactic acid concentration in animal gastrointestinal content. The samples were pre-treated with 25% metaphosphoric acid to remove biological macromolecules including proteins. Then the targeted organic acid concentrations were analyzed using HPLC at column temperature of 60 ℃, with 0.005 mol/L sulfuric acid solution as mobile phase at a flow rate of 0.7 mL/min, and with an Ultraviolet Detectors at the wavelength of 210 nm. Results showed that the determination coefficient (R2 ) of calibration curves based on values of peak area and molar concentration of each organic acid was not less than 0.999 9, suggesting a great linear relationship. The linear concentration range of lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid was 0. 043 9 to 7. 029 4 mmol/L, 0. 022 7 to 9. 076 3 mmol/L, 0.554 7 to 88. 748 0 mmol/L, 0. 510 1 to 81. 622 8 mmol/L, 0. 041 3 to 6. 606 2 mmol/L, 0. 167 3 to 26.763 7 mmol/L, 0.035 6 to 5.699 8 mmol/L and 0.044 5 to 7.121 8 mmol/L, respectively, and the detection concentration limits of the organic acids ranged from 0.005 8 to 0.194 1 mmol/L. The relative standard deviation (RSD) of the peak area of each organic acid within 72 h in standard mix was less than 3.30% while the standard deviation of retention time of each organic acid was less than 0. 056 min, suggesting a good stability within 3 days. The RSD of peak area of each organic acid in employed rumen fluid sample was less than 3.20%, and the recovery rates of each organic acid at three different concentrations of standards were 80.562 8% to 111.496 0%, suggesting that the method has a good accuracy and precision. In conclusion, the method developed in this study has low requirements for instrument configuration, relatively simple pre-treatment strategy and mobile phases, good system adaptability, linearity, and stability, and high precision and accuracy, which can provide technical support for the study of nutrient digestion and absorption, methanogenesis and metabolic diseases in animal gastrointestinal tract.