An aqueous two-phase system (ATPS) containing polyethylene glycol (PEG) and potassium phosphate (K2HPO4) was used for partitioning and partial purification of alkaline protease produced by Bacillus licheniformis ATCC 21424. The effect of PEG molar mass, pH and concentration of NaCl salt addition on partition coefficient, yield and purification factor (PF) for ATPS were studied. The highest partition coefficient (5.3) was achieved in an ATPS of 25% (w/w) PEG 10,000 (g/mol) and 10% (w/w) NaCl at pH 9. Cation and anion exchange chromatography was employed for purification of alkaline protease from protein mixture and the yield and PF for enzyme fractions were obtained to make a comparison between ATPS and ion exchange chromatography. Although, the results of the study indicated that using ATPS leads to lower PF than ion-exchange chromatography, but considering its simplicity and high yield adding up lower investments make ATPS a promising purification option in industrial scale. (C) 2012 Elsevier B.V. All rights reserved.