Methods are disclosed for sterilizing preparations of digestive enzymes to reduce the level of one or more active biological contaminants or pathogens therein, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and/or single or multicellular parasites. These methods involve sterilizing preparations of digestive enzymes, such as trypsin, α-galactosidase and iduronate-2-sulfatase, with irradiation.