This work presents the development and validation of a kinetic model describing the enzymatic hydrolysis of a specifically designed fluorogenic probe for free Penicillin-G Acylase (PGA). The model construction involved tracking reaction kinetics through UV–Vis spectroscopy, identifying product-induced inhibitory effects, and employing initial velocity analysis alongside parameter estimation techniques. The kinetic model was structured around a simple ordered uni-bi mechanism comprising three reversible reaction steps. Validation of the model was performed through spectrofluorometric measurements, successfully predicting the fluorescence intensity progression resulting from the enzymatic cleavage of the probe.