The use of ionic liquids (ILs) as a modifying agent of proteins has opened up new opportunities to improve some biochemical properties of enzymes and consequently the biotechnological processes that involve them. In this study, the commercial protease Alcalase was pretreated with the IL tetramethylammonium bromide (1M) and the optimum pH and temperature were investigated using an experimental design with 11 assays. The biochemical properties of the non-treated enzyme (control assay) were also determined to a comparative analysis. The results showed that the effect of the IL on Alcalase® 2.4L properties was strongly affected by the pH and temperature conditions, being able to generate increases or reductions in the enzymatic activity. The most prominent and positive effects of the IL were detected at pH 7-9 and temperatures ranging from 33 to 40°C, in which increases above 80% in protease activities were observed. The IL presented a negative effect at pH 8 and 67°C, reducing the enzyme activity by 78% when compared to the control assay. According to the statistical analysis of the experimental design, the critical values for maximum protease activity of the non-treated Alcalase® 2.4L were pH 8.15 and 53.7°C, with a predicted value of 284,705 U mL-1. On the other hand, the pretreated enzyme showed a strong shift of the optimum pH from 8.15 to 11.9, with the maintenance of the optimum temperature around 53°C. In these conditions, the predicted value of protease activity would reach 403,529 U mL-1. An analysis of variance (ANOVA) showed coefficients of determination (R²) of 0.86 and 0.88 for the non-treated and treated enzyme, respectively, indicating that the models can explain 86-88% of the variability in the experimental data. The values obtained in the F test for the equations were higher than the critical values, indicating the significance of the models.