Abstract Papain thermostability was studied, and non-first-order deactivation kinetics were observed. The results obtained were analyzed by a two-step series-type deactivation model involving the native and active enzyme, an active intermediate enzyme state, and a final inactive state, with excellent agreement. The influence of different polyhydroxylic cosolvents (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of papain at 60°C was also studied. Analysis of the results by the assayed model showed that the main protective effect of cosolvents was observed in the second step of the deactivation profile. The results obtained were analyzed as a function of both the thermodynamic parameters and a protective effect, defined as the ratio of papain half-lives (with and without cosolvents) for the second deactivation step, showing in both cases an important stabilizing effect of these cosolvents on the enzyme. The overall protective effect of cosolvents was also related simultaneously to their concentration and their water activity-depressing power.