OBJECTIVE To establish an HPLC method for determination of related substances in dimercaptosuccinic acid. METHODS An Ultimate XB-C₁₈（4.6 mm×150 mm，5 μm） column was applied. Mobile phase A was tetrabutylammonium bisulfate phosphate solution containing tetrabutylammonium bisulfate 3.2 g·L-1， potassium dihydrogen phosphate 6.5 g·L-1 and disodium edenate 0.09 g·L-1， mobile phase B was meathanol. Gradient elution program： 0 min，100%A；10 min，100%A；20 min，80%A；50 min，80%A. The flow rate was 1.0 mL·min-1. Detection wavelength was set at 220 nm. RESULTS The linear range of dimercaptosuccinic acid was 14.0-112.0 μg·mL-1 （r=0.999 3），limit of detection was 1.8 μg·mL-1. The linear range of acetylenedicarboxylic acid was 10.3-82.3 μg·mL-1 （r=1.000 0），limit of detection was 0.4 μg·mL-1，while the average recovery was 98.3%. CONCLUSION This method proved to be specific，accurate and suitable for determining the related substances in dimercaptosuccinic acid.