Abstract: The application of high-performance liquid chromatography (HPLC) in the separation and quantification of isomers (meta- and ortho-) of p-toluenesulfonic acid was explored. The optimum separation condition was achieved on a Hypercarb (3 μm, 100 mm×4.6 mm) column by optimising the mobile phase (0.1% perchloric acid/acetonitrile), column temperature (30 ℃) and detection wavelength (210 nm). Under the gradient elution mode, the efficient separation of meta-, and ortho-isomers of p-toluenesulfonic acid were successfully achieved, with the resolution exceeding 1.5. Furthermore, the standard curves were constructed based on external standard method, which confirmed that meta-, and ortho-toluenesulfonic acid had good linear correlations in the concentration ranges of 0.056~1.12 mg/mL and 0.054~1.09 mg/mL, respectively. In addition, through the validation test with spiked recovery, the recoveries of the isomers were in the range of 96%~104%, and the relative standard deviations (RSDs) were 0.32%~2.02%, which fully proved the accuracy and reliability of the method in quantitative analysis.