The molecular interactions between HSA and Testosterone have been successfully investigated. The absorption, distribution and metabolism of many molecules can be altered based on their affinity to HSA. HSA is often increases the apparent solubility of hydrophobic ligands in plasma and modulate their delivery to cells. In this study, the interaction between Testosterone and HSA has been investigated using UV-VIS absorption spectrophotometry and FT-IR spectroscopy; binding constant and the effects on the protein secondary structure have been confirmed. From UV-VIS absorption spectrophotometry which showed an increase in the absorption intensity with increasing the molecular ratios of testosterone to HSA, it is found that the value of the binding constant of testosterone to HSA, K equals 34.9×102 M-1. FT-IR spectroscopy in the mid infrared region with Fourier self deconvolution, second derivative, difference spectra, peak picking and curve fitting were used to determine the effect of Testosterone on the protein secondary structure in the amides I, II and III regions. From the FTIR absorbance spectra, it is found that the intensity of the absorption bands increased with increasing the molecular ratios of Testosterone, where from the deconvoluted and curve fitted spectra, it is found that the absorbance intensity for α- helices decreases relative to β- sheets; this decrease in intensity is related to the formation of H- bonding in the complex molecules.