Characterization of total proteases, trypsin and chymotrypsin were performed at pH 2 12 andtemperatures 20-80°C in three life stages of Siamese fighting fish (Betta splendens Regan, 1910). Atleast fourteen protease activities were detected, including seven acidic (pH levels 2, 4 and 5-6), oneneutral (pH 7), and six alkaline (pH levels 8-12) activities in the optimal temperature range of 30-60°C.The neutral and alkaline pH profiles were different between sexes during the on-growing stage (1.5month-old). The acidic proteases played a major role in digestion in the early life stage (10-day-old),while the alkaline proteases became more important toward older stages. Trypsin and chymotrypsinshowed similar characteristics with at least five activities observed. For trypsin, activity was detectedwith pH 7 and 8 at 50°C, and pH 10 at 30, 50 and 60°C. For chymotrypsin, activity was found at pHlevels 7 and 8 at 50°C, and pH 10 at 30, 40 and 50°C. Regardless of sex and age, the most suitableconditions for studying digestive enzyme activities in general were at pH 8 and 50°C for total proteases,pH 7-8 and 50°C or pH 10 and 30-35°C for trypsin and pH 7-8 and 50°C or pH 10 and 40°C forchymotrypsin. Trypsin and chymotrypsin are important proteases showing similar general optimalconditions to that of total proteases. This information provides elementary knowledge for studyingprotein digestibility by in vitro techniques and for determining growth performance quality using digestiveenzyme markers in Siamese fighting fish. Journal homepage: http://kasetsartjnatsci.kasetsart.org/