Abstract: OBJECTIVE To establish an HPLC method for determination of related substances in Azathioprine Tablets. METHODS The C₁₈ column (200 mm×4.6 mm, 5 μm) was used. The mobile phase consisted of methanol and 0.05% of sodium acetate solution(18∶82) at the flow rate of 1.2 mL·min^-1, and the detection wavelength was set at 300 nm. RESULTS The linear ranges of the two typical related substances mercaptopuring and 5-chloro-1-methyl-4-nitroimidazole in Azathioprine Tablets were all 0.125-1.80 μg·mL^-1(r=0.9997 and 0.9999) with the detection limits of 0.787 ng and 0.933 ng, and the average recoveries were 100.3% and 100.0% with the RSD of 0.64% and 0.14%. CONCLUSION The method proposed for determining the related substances of Azathioprine Tablets is simple, quick and the result is accurate and specific.