The objectives of the present study were to characterize biochemically the proteases present in gastric juices from adults of the southern king crab (Lithodes santolla) and the Chilean rock crab (Cancer edwardsii), to determine the classes of proteases using specific inhibitors, as well as to evaluate the effect of temperature and pH that affect the proteolytic activity. The enzyme activity analysis revealed non-significant differences between the activities of serine proteases (trypsin and chymotrypsin) in both species. On the other hand, acid protease activity in C. edwardsii was significantly higher than in L. santolla. The activity of aspartate proteases was inhibited by Pepstatin A in both species, with no significant differences in the extent of inhibition between them. The maximal enzyme activity detected for both species occurred at alkaline pH. The optimum pH for proteolytic activity was ranged between 7 and 9 for C. edwardsii, and between 7 and 11 for L. santolla. Moreover, the highest proteolytic activity was recorded at 60°C in both species. However, at this temperature, enzyme activity was unstable after 20 min. Finally, collagenase-like activity in the gastric juices was detected for both species; this activity deserves further investigation, considering its biotechnological potential.