Photophysical studies were carried out for simple amino acids like alanine and valine with resorcinol-based aqueous acridinedione (ADDR) dyes. ADDR dyes exhibit interesting excited-state characteristics on altering the substituents at the 9th and 10th sites (Scheme 1). The longest-wavelength absorption maxima remain the same on adding the amino acids to the fluorophore, whereas the excited-state behavior varies significantly mostly based on the nature of the substituent at the 9th position. The absence of fluorescence enhancement was observed with addition of β-alanine, l-alanine, and l-valine to ADDR1 dye (photoinduced electron transfer, PET), whereas addition of glycine exhibits enhancement accompanied with a shift toward a longer-wavelength region. Interestingly, the addition of amino acids to non-PET dyes results in a fluorescence quenching accompanied with a larger shift toward the shorter-wavelength region. The properties of fluorophore and nonfluorophore dyes in the presence of alanine or valine are found to be entirely different from those of glycine. The interaction of alanine with ADDR dyes is predominantly through H-bonding, but the structural aspects of H-bonding interactions of alanine and water are completely different from those of glycine and water. The time-correlated single-photon counting method portrays the existence of fluorophore in two distinguishable microenvironments in the presence of amino acids. The fluorescence spectral technique used as a tool in elucidating the mode of interaction of dye with neutral amino acids in aqueous solution is illustrated in the present study.