A novel approach on fluorescence quenching of tyrosine and l-tryptophan is presented for spectrofluorimetric determination of aniracetam in drug substances and products. The quenching mechanism was investigated using Stern-Volmer plots and ultraviolet spectra figures of quencher-fluorophore mixtures. Binding constant and stoichiometry were calculated using double-log plots. The spectrofluorimetric method was optimized for the experimental conditions affecting fluorescence quenching including fluorophore concentration, diluent, and reaction time. Moreover, the pH-rate profile of aniracetam was studied using simple kinetics and found to be stable within the pH range 5-8. Fluorescence quenching of tyrosine and l-tryptophan were observed on addition of aniracetam in aqueous medium at pH 5.5-6.5. Aniracetam quenched the fluorescence of tyrosine and l-tryptophan in the concentration range 1-20 μg/ml and 0.3-20 μg/ml, respectively, with binomial relationships between quenching values (ΔF) and aniracetam concentration. Limits of detection were found to be 0.10 μg/ml for tyrosine-aniracetam and 0.14 μg/ml for l-tryptophan-aniracetam. Method validation was performed as per ICH guidelines and demonstrated that the developed spectrofluorimetric method was accurate, precise, specific, and suitable for analysis of aniracetam in routine quality control laboratories. All experimental materials and solvents used are eco-friendly, indicating that the cited spectrofluorimetric procedure is an excellent green method.