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Azo dyes are made in significant amounts annually and released into the environment after being employed in the industry. There are some reports about the toxic effects of these dyes on several organisms. Thus, the textile dye Reactive Black 5 (RB5) has been examined for its cytotoxic effects on the human serum albumin (HSA) structure. Molecular interaction between RB5 and HSA indicated the combination of docking methods, molecular dynamic simulation, and multi-spectroscopic approaches. HSA's intrinsic fluorescence was well quenched wi