The application of aqueous two-phase systems (ATPS) is a cost-effective and simple method of protein separation (including enzymes) from complex systems. The first stage of designing the protein purification process in an ATPS involves the identification of the conditions for the formation of a given extraction system. For this purpose, the conditions for the formation of ATPSs in a thermoseparating EO50PO50 polymer/potassium phosphates system have been studied. Factors determining the ATPS formation comprised: separation temperature (4ºC or 20ºC), phosphate solution pH (6, 7.5 or 9) as well as the concentration of NaCl introduced into the systems (0.085 M, 0.475 M and 0.85 M). ATPS without NaCl were prepared as well. The conditions for the formation of the primary EO50PO50/potassium phosphate ATPS were determined with their phase diagrams. It was observed that with an increase of phosphate pH and NaCl concentration in the system, there was a decrease of the EO50PO50 and phosphate concentrations necessary to form a primary ATPS. After the primary two-phase separation, the top phase (rich in the EO50PO50 polymer) was partitioned from the bottom phase (rich in phosphates). Next, by means of polymer phase thermoseparation, a secondary two-phase system was formed. In the secondary EO50PO50/phosphate ATPS, the bottom phase was formed by the concentrated EO50PO50 polymer (30-80% concentration), while the top phase by a solution composed mainly of water, containing phosphate ions and remains of EO50PO50 polymer (3-7%).