Losartan potassium (LP) is an antihypertensive drug, and its binding to plasma proteins determines its pharmacokinetic properties. The presence of an exogenous substance like Caffeine (CAF) may affect the binding of drugs to proteins. Accordingly, we aim to characterize LP and bovine serum albumin (BSA) interactions in CAF presence employing spectroscopic techniques (UV-vis) absorption and fluorescence measurements) under physiological circumstances. Our findings indicated that the quenching effect of LP on BSA was decreased by adding CAF by calculating the Stern-Volmer constant. Hence, the Modified Stern-Volmer equation was utilized for calculating binding constants for the BSA-LP and BSA-CAF-LP systems, and the values were 4.47×103and 0.23×103, respectively. Thermodynamic parameters, the Gibbs free energy (∆G), enthalpy (∆H), and entropy (∆S) calculated for each system show spontaneous interaction for both systems and suggest that the binding interaction between BSA and LP is exothermic and entropy is unfavorable, while for BSA-CAF-LP is endothermic and entropy is favorable.