According to research, thiosalicylic acid has anti-inflammatory, antioxidant, and analgesic effects. Additionally, some of its derivatives have shown significant antimicrobial and antitumor activity. In vitro studies have also shown that S-alkyl derivatives of thiosalicylic acid exhibit moderate and dose-dependent cytotoxic effects on human colon and lung carcinoma cells. In this reserach, we utilized various spectroscopic methods and molecular docking simulation to examine the binding interaction between human serum albumin (HSA) and potential biologically active isopropyl derivatives of thiosalicylic acid (ligand, L). To analyze the quenching mechanism, the association constants and number of binding sites were utilized based on the obtained results. The tested L and HSA had a static fluorescence quenching mechanism, while their binding processes were spontaneous. Additionally, UV-Vis absorption spectroscopy revealed that the binding of the tested L induced slight conformational changes in HSA.