Developing a novel Ionic-liquid (IL) based aqueous two-phase system (ATPS) with polyethylene glycol (PEG) as adjuvant for the separation of biomolecules is studied. This original work involves addition of various concentration of PEG (2000, 4000, and 6000 gr/mol) to 1-butyl-3-methylimidazolium acetate + potassium hydrogen phosphate ATPS to investigate their subsequent effect on phase diagrams and partitioning coefficient of a-amylase. In another innovative aspect of this work, response surface methodology (RSM) based on three-variable central composite design was employed to understand the effect of phase forming components on extraction studies of a-amylase. The addition of small amount of PEG improved the partitioning coefficient of biomolecule. The effective excluded volume theory was applied to correlate the salting-out ability. As a result, it can be stated that the proposed system can effectively be used in separation and purification studies instead of task specific ILs. (C) 2015 American Institute of Chemical Engineers