AbstractKinetic parameters of operational inactivation of horseradish peroxidase during the enzymatic oxidation of 2‐aminophenol with hydrogen peroxide were estimated using an isoconversional method. This method only requires the progress kinetic curves obtained at constant initial concentrations of the substrates. In order to investigate the possible pathways of low‐reactive species compound III formation, a kinetic model was proposed and several rate constants were calculated using an optimization computing program. Generation of an oxygen reactive species in the reaction system was also considered in the reaction scheme. Sensitivity analysis, corroborated with the overall inactivation kinetics observed with the isoconversional method, allows the conclusion that the inactivation of the enzyme occurs mainly via the reaction of compound II with hydrogen peroxide.