Proteolytic enzymes have various applications in biomedicine, biotechnology, pharmaceuticals, and the food industry. Some of these applications require incubation at extreme temperatures, high pressure, different pH of the solution, and the presence of various additives that might destabilize enzyme structure and function. Structural features of proteases define the mechanism of denaturation, which is reflected in different activation energies for the process. Understanding the activation energy gives clues about the kinetic inertness of the enzyme and its resistance to destabilizing factors. Monitoring the kinetics of enzyme thermal inactivation by measuring activity loss upon heating at various temperatures enables the determination of activation energy for the inactivation process using the Arrhenius plot. This way, measuring the activity loss as a result of structural perturbations gives direct information on the enzyme stability in the relevant conditions for applicative processes.