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The molecular interactions between HSA, Testosterone and Progesterone have been successfully investigated. In this study, the interaction between Testosterone, Progesterone and HSA has been investigated using UV-VIS absorption spectrophotometry. From UV absorption spectrophotometry it was shown that the absorption intensity decreasing with increasing the molecular ratios of Progesterone-HSA, while the absorption intensity increases with increasing the molecular ratios of Testosterone-HSA For VIS absorption spectrophotometer it was shown a decreasing in the absorption intensity with increasing the molecular ratios for both Progesterone and Testosterone. From both the UV-VIS spectrophotometer studies, is found that the value of the binding constant of testosterone to HSA, K equals 34.9×102 M-1 , While the binding constant (k) for the Progesterone is estimated as 6.56×102 M-1 at 293 K. From the results, it was observed that strong binding of the testosterone than that of progesterone.