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A simulation procedure for the optimization of enzymatic reactors used in sandwich flow injection systems is evaluated. The system is modeled as a plug‐flow reactor with axial dispersion. To calibrate it, dispersion coefficients can be evaluated using residence time distribution techniques; meanwhile, enzymatic kinetics must be determined for the system considered, according to the values of the substrate conversion attained. The model has been linked to an optimization routine based on the Powell algorithm. The proposed approach has been evaluated in a system performing simultaneous determinations of glucose and glycerol, considered the common carbon sources in a fermentation process.