Partitioning and recovery of proteases from stomach extract (SE) and acidified stomach extract (ASE) of lizardfish using a three-phase partitioning (TPP) system in combination with an aqueous two-phase system (ATPS) was optimized. The highest yield and purity were obtained in the interphase of the TPP system, which consisted of a SE or ASE tot-butanol ratio of 1.0 : 0.5 in the presence of 40% (w/w) (NH4)2SO4. Both TPP fractions were further subjected to ATPS. Phase compositions of ATPS including PEG molecular mass and concentrations as well as types and concentrations of salts influenced protein partitioning. The best ATPS conditions for protease partitioning into the top phase from TPP fractions of SE and ASE were 15% Na3C6H5O7–20% PEG1000 and 20% Na3C6H5O7–15% PEG1000, which increased the purity by 4-fold and 5-fold with the recovered activity of 82% and 77%, respectively. ATPS fractions of SE and ASE were subsequently mixed with several PEGs and salts for back extraction (BE). BE using 25% PEG8000–5% Na3C6H5O7gave the highest PF and yield for both ATPS fractions. SDS-PAGE investigation revealed that the decrease in contaminating protein bands was observed after the combined partitioning systems. BE fractions of SE and ASE were quite stable at −20 and 0 °C up to 14 days. Therefore, the combination of TPP, ATPS and BE could be effectively applied to recover and purify proteases from the stomach of lizardfish.