Aqueous two phase systems (ATPSs) formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00 are non conventional systems of low impact. Their ability to purify trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) from bovine pancreas was evaluated. The effect of several variables such as molecular weight of PEG, tie line length and phase-volume ratio on zymogen partitioning was explored. Those systems formed by PEG of molecular weight 600 (20.62%, w/w) and NaTart (12.85%, w/w) with a phase-volume ratio equal to one were found to evidence the best purification performance, and were employed to design a two-stage purification process. Firstly, a ATPS extraction step yields both to recover most of pancreatic TRPz and ChTRPz (76% and 87% respectively) in the PEG600 enriched-top phase and to eliminate other pancreatic homogenate components - cell debris, nucleic acids and lipids - which either partition into the bottom phase or precipitate at the interface. Then, the top phase - from this first extraction step - is mixed with a given quantity of PEG6000 in order to induce the formation of a new aqueous two phase system in which the back extraction of both zymogens takes place. This second stage allows to obtain a mixture of both zymogens highly pure (congruent to 95%) in a bottom phase with low PEG concentration (3.70%, w/w) and therefore suitable for being used in multiple applications. (c) 2011 Elsevier B.V. All rights reserved.