Abstract The performance of the standardized extrusion-spheronization technique, operational conditions, formulation parameters and storage of the final product over the bioactivity of papain containing pellets has been evaluated to obtain an insight into the potential of the technique for the manufacture of solid protein formulations. The pellets produced were assayed in terms of biological activity - monitored at each operational stage using N-benzoyl-dl-arginine ρ-nitroanilide as a substrate, and according to the physical properties - evaluated by means of size distribution, apparent density and friability. The produced pellets presented adequate physical and mechanical properties. Monitoring biological activity at each production stage revealed that the most critical steps corresponded to drying and storage, with bioactivity decay ranging from 5 to 30% and 5 to 20% for each process. Dry mixing and extrusion did not hold any influence over papain activity, while wet massing decreased the bioactivity by approximately 0-5% and the spheronization 0-2%. The results varied as a function of the experimental conditions and formulation components. In conclusion, the extrusion--spheronization technique was suitable to produce solid multiparticulate dosage forms for papain, considering the possibility to originate pellets with relatively low bioactivity decay. However, weak points of the technique corresponded to the wet massing and drying stages as well as storage.