Abstract <p>This study aims to investigate the interaction of olmesartan drug with human serum albumin using fluorescence, circular dichroism spectra and molecular docking techniques under physiological conditions. Fluorescence quenching of human serum albumin by olmesartan indicated that a moderate binding affinity (<i>K</i>_a = 3305 M^–1) and spontaneous reaction between olmesartan and HSA obtained in phosphate buffer (0.05 M) and pH 7.4 at 25°C. The dichroism spectra results revealed a decrease in the α-helical content of human serum albumin from 61.1 to 59.2% with the addition of olmesartan, indicating that olmesartan binding induces changes in the secondary structure of human serum albumin. The study of molecular docking also indicated that the optimal binding site for olmesartan on human serum albumin is located in the IIA and IIB subdomains. Thermodynamic analysis and molecular docking results suggested that the binding of olmesartan to human serum albumin is dominated by hydrophobic interactions and hydrogen bonds. Also, olmesartan formed hydrophobic interactions with Trp214, Asp451, Tyr452, and Asn295, and established five hydrogen bonds with Lys195, Arg218, and Pro339. However, theoretical and experimental findings demonstrated excellent agreement.</p>