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The research examines, numerically and experimentally, the identification of substrate concentration in amperometric electrochemical flow cells. Three-dimensional numerical simulations have been preformed for predicting the mass transfer processes in the vicinity of the electrochemical cell using a realistic geometrical model. The experimental procedure included the fixed enzyme β-galactosidase and the injected substrate, para-aminophenyl β-D-galactopyranoside. For the optimization of the inlet flow rate, simulations and experiments have been preformed using flow rates between 0.05-250 μl/min with an identical substrate concentration. The numerical simulation results were used to evaluate the species concentration distribution in the vicinity of the electrochemical cell for predicting the electric current through the electrode. Different substrate concentrations applied and ranged between 0.05-1.125 mg/ml at a chosen flow rate of 50 μl/min. A good agreement was found between the numerical and the experimental electric current evolution values, especially for the higher substrate concentrations. The correlation coefficient was 0.98 in the higher substrate concentrations. A linear relationship was obtained between the inlet substrate concentration and the steady-state electric current for both the numerical and the experimental results. Once this linear relationship is established, the inlet substrate concentration based on the electric current through the electrode can be established.