The present study aimed to characterize chymotrypsi n-like enzymes in three species, namely the mudcrab Scylla serrata, the brine shrimp Artemia salina and the ro tifer Brachionus plicatilis. Optimized conditions of assay were established in terms of the volume of crude extract s used and the time of reaction. The crab enzyme wa s the most affected by pH showing big increases in its activit y until pH 8.0 and abruptly decreased beyond this l evel. The Artemia chymotrypsin exhibited maximal activity at 7.0 - 7.5 while the rotifer enzyme was the least af fected by pH with small increases in its activity until its maxi mum level at pH 8.5. The stability of the mud crab enzyme was the most affected by pH; the Artemia and the rotifer en zymes exhibited maximal activity at 7.0 to 7.5. Th e enzyme activity of the mud crab was maximal at 30 o C and decreased abruptly at higher temperature. In contrast, the Artemia chymotrypsin-like activity was practically unaffected by temperature and the rotifer enzyme ex hibited maximal activity at 25 o C and gradually decreased with increased temperatur e.Thermal stabilities were slightly affected in all three species;a small peak was obse rved in the mud crab enzyme at 25 o C. The rotifer and the Artemiaenzyme stabilitydecreased slightly and linea rly with temperature. The determined Km for benzoy l-L- tyrosine ethyl ester (BTEE) of the rotifer, Artemia , and the crab chymotrypsin-like enzymes were estim ated to be 1.3, 0.4 and 0.5 nmol N-benzoyl-L-tyrosine produced min -1 mg protein -1 , respectively. Conclusion: Chymotrypsin-like enzymewas stable at alkaline pH and at room tempera ture or above for all three species. The Artemia a nd the mud crab chymotrypsin-like activity manifested higher s ubstrate-enzyme affinity in contrast with the rotif er enzyme which exhibited the least affinity.