Abstract: A HPLC method for analysis of gossypol using reverse phase C₁₈ column (ODS), solvent system THF-water (60:40 v/v) with 0.08% phosphoric acid and detection at 254 nm, was developed.Compared with methods reported in the literature, this method exhibits higher separative and more stable effect. It gives a nice sharp peak for gossypol, while the impurities not observed by other reported methods are well separated in this system.The method was compared with spectrophotometric and thin layer chromatographic procedures currently used for gossypol determination and detection.