Abstract: OBJECTIVE To establish an HPLC method for content determination of clioquinol and related substances in clioquinol raw material. METHODS A C8 column was used with the mobile phase of the buffer solution-methanol (35∶65). The flow rate was 1.0 mL·min-1 and the detection wavelength was 254 nm. RESULTS The calibration curves of clioquinol was linear in the range of 3-60 μg·mL-1(r=0.999 9), and the average recoveries was 99.8%, RSD was 0.13%(n=9). CONCLUSION The method is rapid, accurate and reliable for determining the content and related substances in clioquinol raw material.