Nile red and Nile blue are intensely colored fluorescent dyes from the benzophenoxazine family. Modification of the donor amine with varying alkyl substituents was achieved with the principle aim being to investigate differences, both optical and physical, between dye scaffolds outfitted with secondary amines versus tertiary amines. These changes to the dyes’ architectural framework gave holistic effects to the bulk molecule by modulating hydrophobicity, optical properties, and protein binding constants to human serum albumin.