Proteases are widely used in industrial processes, and the discovery of new, more kinetically efficient proteases can have a positive impact on industry. Enzymes from Antarctic microorganisms exhibit cold-adaptive properties, making them useful in biotechnology. The cold and harsh environment of Antarctica makes it a valuable source for new biotechnologically related enzymes. In this study, we characterized two cold-adapted proteases purified from Pseudoalteromonas issachenkonii P14M1-4 and Flavobacterium frigidimaris ANT34-7, isolated from King George Island, Antarctica, and compared these with proteases from the non-cold-adapted bacteria Bacillus licheniformis and Geobacillus stearothermophilus. The best temperature growing conditions were used for protease purification and characterization. The protease from P. issachenkonii P14M1-4 was identified as a 40–43 kDa metal-dependent subtilisin-like serine protease and the protease from F. frigidimaris ANT34-7 was identified as a 28 kDa metalloprotease. The enzymes showed an optimum temperature of between 35°C and 40°C and an optimum pH in the neutral to alkaline range. Their activation energies, catalytic constants and growth capacities at different temperatures categorize them as cold-adapted enzymes. We conclude that the characteristics exhibited by these proteases make them useful for biotechnological purposes requiring high activity at low temperatures. Moreover, to the best of our knowledge, this is the first characterization of a cold-adapted protease from F. frigidimaris.