Ionic liquids (ILs) have shown great potential as solvent media for biotechnological processes, such as those involving enzymatic reactions. It has been demonstrated that these compounds can alter the physiochemical properties of proteins, improving the activity and stability of enzymes. Thus this work evaluated the effects of ILs on the kinetic parameters of proteolytic enzymes. The commercial proteases Alcalase® 2.4L, Flavourzyme® 500L and Neutrase® 0.8L and the ILs choline chloride and tetramethylammonium bromide were used in this study. The kinetic parameters of activation energy (Ea), the Michaelis–Menten constant (Km), maximum velocity (Vmax), the half-life of the enzyme (t1/2) and the decimal reduction time (D value) were comparatively analyzed for treatments in the presence and absence (control) of ILs, and the results were expressed in terms of percentage. The combination of tetramethylammonium bromide (2000 mM) and Alcalase® 2.4L resulted in a 50% reduction in the parameter Ea, and 3-fold and 2-fold increases in Km and Vmax, respectively, as compared to the control sample. Alcalase® 2.4L was also more thermally stable in the presence of the IL, presenting 16-fold greater values for t1/2 and D than the control assay. For the protease Neutrase® 0.8L treated with choline chloride (0.5 mM), no significant changes were detected for the Ea, t1/2 and D values, although the parameter Km showed a decrease of 18%. The combination of Flavourzyme® 500L and choline chloride (5 mM) resulted in a 19% reduction in Km and 2-fold higher values for t1/2 and D as compared to the control assay. In summary, these results allow for the conclusion that the use of ILs in enzymatic reactions can promote significant changes in the kinetic parameters of proteases. The most positive effects observed in this study included: proteases with improved activity and stability properties as well as greater affinity for the substrate.