This study investigates the non-native interactions between the photosensitizer protoporphyrin IX (PPIX) and human serum albumin (HSA). Non-covalent binding between small molecules and proteins, is crucial for various applications in biomedicine, food processing, energy conversion, and sensing. The research focuses on the role of a series of organic solvents in facilitating the binding of water-insoluble PPIX to the protein. By using dialysis and centrifugation for sample preparation and combining experimental and computational methods for characterization, the study found that non-protic solvents such as THF and DMSO are more effective in forming the PPIX:HSA complex compared to protic solvents. Additionally, the temporary presence of these organic solvents during incubation does not cause significant and irreversible changes in the protein structure. Instead, THF and DMSO temporarily loosen the protein, increasing the distance between two tyrosine residues (Y138 and Y161) that are believed to coordinate the porphyrin at its binding site. This finding underscores the importance of selecting appropriate solvents to enhance the binding efficiency of small ligands to proteins.