The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome activity assays, represented by the trypsin-like, chymotrypsin-like or caspase-like site, lies in the fact that most commonly used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we showed that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20-60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.