A novel approach of using near infrared region (NIR) dyes is applied to elucidate the binding interaction between human serum albumin (HSA) and mangiferin (MGF). HSA is a blood carrier protein used for drug delivery, while mangiferin is a natural polyphenol found in mangoes that possesses numerous beneficial health properties. The NIR dyes are used as a probe to investigate MGF binding interaction with HSA via monitoring fluorescence of Trp-214 residue. Molecular modeling is used for docking and semi-empirical analysis. The investigation of the binding interaction between Trp-214 and MGF is significant, for it may offer broader pharmacological insight and applications for the polyphenol. Mangiferin in proposed to bind with a 2:1 stoichiometric ratio with HSA to the Trp-214 residue in subdomain IIA and another possible binding site to be determined in future studies. Spectral changes suggest a stabilized protein conformation upon mangiferin binding with the addition of NIR dye E-06 and MHI-06.