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This study aims to demonstrate the benefits of augmenting commercially available, real‐time, in vitro glycolysis assays with phenomenological rate equation‐based kinetic models, describing the contributions of the underpinning metabolic pathways. To this end, a commercially available glycolysis assay, sensitive to changes in extracellular acidification (extracellular pH), was used to derive the glycolysis pathway kinetics. The pathway was numerically modelled using a series of ordinary differential rate equations, to simulate the obtained experimental results. The sensitivity of the model to the key equation parameters was also explored. The cellular glycolysis pathway kinetics were determined for three different cell‐lines, under nonmodulated and modulated conditions. Over the timescale studied, the assay demonstrated a two‐phase metabolic response, representing the differential kinetics of glycolysis pathway rate as a function of time, and this behaviour was faithfully reproduced by the model simulations. The model enabled quantitative comparison of the pathway kinetics of three cell lines, and also the modulating effect of two known drugs. Moreover, the modelling tool allows the subtle differences between different cell lines to be better elucidated and also allows augmentation of the assay sensitivity. A simplistic numerical model can faithfully reproduce the differential pathway kinetics for three different cell lines, with and without pathway‐modulating drugs, and furthermore provides insights into the cellular metabolism by elucidating the underlying mechanisms leading to the pathway end‐product. This study demonstrates that augmenting a relatively simple, real‐time, in vitro assay with a model of the underpinning metabolic pathway provides considerable insights into the observed differences in cellular systems. [ABSTRACT FROM AUTHOR]