BACKGROUND: L-asparaginase (ASNase) is an important biopharmaceutical used to treat the acute lymphoblastic leukemia (ALL) and lymphosarcoma. Considering its main use in cancer therapy, the most important request for ASNase production is the need for a highly pure biopharmaceutical obtained in the final of the downstream process, which is considered as the crucial step in its production. RESULTS: This work proposes the use of polymer–salt aqueous two-phase systems (ATPS) based on polyethylene glycol and citrate buffer, with ionic liquids (ILs) as adjuvants, combined with the permeabilization of cell membrane using n-dodecane and glycine for the in situ purification of periplasmatic ASNase from Escherichia coli cells. The process proposed was optimized (polymer molecular weight, pH, tie-line length/mixture point, presence, nature and concentration of the adjuvant). The results show that ASNase partitions mostly to the PEG-rich phase, due to hydrophobic interactions between both PEG and enzyme. Remarkably, the addition of 5 wt% of 1-butyl-3-methylimidazolium methanesulfonate [C4mim][CH3SO3] as adjuvant lead to high recoveries [87.94 ± 0.03 (%)], purification factors (20.09 ± 0.35), and a final specific activity SA = 3.61 ± 0.38 U mg-1 protein, from a crude enzyme extract with a SA = 0.18 ± 0.05 U mg-1 protein. Moreover, better results were achieved when a prepurification step consisting of an ammonium sulfate precipitation was combined with the optimized ATPS, achieving an increased SA = 22.01 ± 1.36 U mg-1 protein and PF = 173.8. CONCLUSIONS: A novel integrated downstream process was successfully implemented for the in situ purification of ASNase from fermentation broth. © 2017 Society of Chemical Industry.