Thiosalicylic acid is known for its diverse pharmacological properties and its frequent use in various synthetic conversions. The unique structure of thiosalicylic acid permits the incorporation of diverse S-alkyl derivatives, resulting in favorable chemical characteristics for numerous applications. Therefore, affinity for one of the binding sites of human serum albumin (HSA) of isoamyl derivative of thiosalicylic acid (ligand, L), were examined. Binding mode of compound L was determined using fluorescence spectroscopy. Warfarin was used as a marker for Sudlow’s Site I (subdomain IIA), while ibuprofen was used as a marker for Sudlow’s Site II (subdomain IIIA). Obtained values of Ka suggested that investigated compound bind to HSA. Results of site marker competitive experiments showed that the tested L bind to HSA in domain IIA (Site I). The presented results will help to improve the research of the mechanism of the interaction between transport proteins and similar compounds.