Background & Aim:Saponins are well-known secondary metabolites with numerous beneficial pharmacological properties. Since the interactions of drugs with blood constituents, in particular with human serum albumin (HSA) may have a major impact on drug pharmacology, the present study designed to provide a fundamental understanding of the interaction of saponins extracted from Tribulus terrestris (typically utilized in folk medicine) with HSA in detail. Experimental: After extraction and purification of saponins; UV-Vis spectroscopy, fluorescence spectroscopy, and far-UV CD spectroscopy were used to examine the effects of saponins from Tribulus terrestris on HSA. Results: Thin-Layer chromatography confirmed the presence of 3 different saponins. The UV-Vis absorption, fluorescence emission and far-UV CD results displayed that saponinsfrom Tribulus terrestris could form a complex with HSA. The binding constant for the saponins–HSA complex was found to be 13.4×104M-1. The distance r between HSA and Tribulus terrestris saponins was also acquired according to the Förster theory. Recommended applications/industries: Altogether, saponins can bind to HSA and change the secondary and tertiary structure of HSA moderately. The results obtained from this study can help in understanding the pharmacokinetic properties of saponins. Manuscript profile