An aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 4000 and potassium phosphate was explored for the separation of protease produced from Bacillus licheniformis NCIM 2042. The various experimental parameters viz. potassium phosphate, PEG, and NaCl concentration as well as system pH were optimized for higher extraction yield using central composite rotatable design (CCRD) of response surface methodology (RSM). The RSM predicted model delivered 86.80% yield with 1.89 purification factor and 11.65 partition coefficient of protease with ATPS comprising of potassium phosphate 22.36% (w/w), PEG 4000 15.93% (w/w), pH (10.06), and NaCl (1.08 mM). The experimental results of optimized conditions showed partition coefficient of 11.95 with 2.2 purification factor and %yield of 89.75 of protease and close resemblance of both the results validated the predicted model. SDS PAGE analysis showed a protein bands for partially purified protease.