Abstract: To investigate the related substances of tenofovir, 3 related substances were synthesized. RP-HPLC analytical method was developed using Agilent ZORBAX SB-C₁₈ column. The mobile phase composed of methanol and 0. 02 mol/L potassium dihydrogen phosphate. Linear gradient elution was used and the detection wavelength was 260 nm. 3 impurities were found in tenofovir. The established method was accurate and reproducible, and it can be applied for the related substances test in tenofovir.