Snakehead fish are being popular in the Mekong Delta with increasing area, however, this fish is only consumed domestically or exported to Cambodia. Research on the development of snakehead fish products is being considered to improve the economic value of this raw material. In addition, utilized thoroughly the by-products from snakehead fish processing into value-added products were also focused. Previously, the identification of the appropriate viscera components for extracting protease stability and protease activity in frozen storage of raw materials are conducted. The conditions for protease extraction from viscera of snakehead fish (Channa striata) were investigated. The individual factors (solvent composition, pH, ratio of raw materials and solvent) which influenced to protease extraction were initially determined. After that, the response surface methodology (RSM) based on two-variable central composite design (CCD) can be used to model the correlation of extraction temperature and time to protease activity. The results showed that ratio of liver and pancreas in viscera of snakehead fish was 43.20%, intestine 31.46%, stomach 15.34%. The highest activity of protease extract from liver and pancreas of snakehead fish was 13.88±0.03 U/g dry materials, followed by the intestine (5.00±0.22 U/g dry materials) and lowest in stomach (0.58±0.11 U/g dry materials). The optimum activity of protease (as calculated) was 55.61±0.42 U/g dry materials and 7.81±0.10 U/mg protein when using phosphate buffer pH 8.0 to extract protease with the 1:2 (w/v) ratio of sample and solvent; extraction temperature was 46.5°C for 32.7 minutes.