Aqueous Biphasic Systems (ABS) composed of Polymers and cholinium-based Ionic Liquids (ILs) are here propose as a biocompatible alternative for the biopharmaceuticals extraction and purification, particularly the therapeutic enzyme L-asparaginase (ASNase). Different ABS composed of cholinium chloride ([Ch]Cl) and different polymers were evaluated. The polymers used as phase forming agents were polyethylene glycol 600 (PEG-600), polypropylene glycol 400 (PPG-400) and polyethylene glycol dimethyl ether 500 (PEG-DME-500). Their extraction efficiencies (EE%) at different weight compositions were studied by measurement of enzymatic activity of ASNase per volume of each phase relative to initial activity of ASNase added in each systems. The results have shown that all the [Ch]Cl-based ABS can concentrate ASNase in the [Ch]Cl-rich phase in a single-extraction step, with efficiencies higher than 98 %. Next studies will evaluate the purity of the ASNase after the extraction, but these results suggest that [Ch]Cl/polymer ABS are efficient and promising platforms for protein separation.