Diabetes management has increasingly relied on dipeptidyl peptidase-4 inhibitors like linagliptin, creating a need for environmentally sustainable analytical methods to replace conventional chromatographic techniques that often involve complex sample preparation, organic solvent usage, and expensive instrumentation. A sensitive and selective "turn-off" fluorescence quenching method was developed and validated for the determination of linagliptin using eosin Y as the fluorescent probe. The spectral characteristics and sensing mechanisms were investigated using Stern-Volmer analysis, Job's method, and thermodynamic studies, revealing a static quenching process driven by the formation of a non-fluorescent 1 : 1 linagliptin-eosin Y complex with a high Stern-Volmer constant (