The increasing demands of consumers cause the manufacturers of food to be more and more interested in replacing commonly used chemical preservatives with natural antimicrobial substances. Among various natural antimicrobial substances, the lysozyme is characterized by properties inhibiting the growth of many pathogenic bacteria, such as Staphylococcus and Streptococcus. Different methods can be applied to separate lysozyme from hen egg white in order to obtain its commercial preparations; however, only a few have been already implemented in the industrial-scale production. They are: repeated salt precipitation and crystallization, direct ultrafiltration and ion-exchange chromatography. Recently, in the expert literature, information has been published referring to researches into the lysozyme extraction from hen egg white using aqueous two-phase systems (ATPS) such as polyethylene glycol (PEG)/salt solution. As compared to other separation and purification techniques of egg white, the application of aqueous two-phase systems is characterized by the following: short time of separation process, low energy consumption, the extraction process progresses under the conditions causing no denaturation of proteins, and a possibility to relatively easily scale-up the entire process. However, in the system composed of polyethylene glycol (PEG) and salt solution, the lysozyme preferentially migrates to the PEG-rich top-phase, and, therefore, it is difficult to subsequently recover this enzyme. The process of separating lysozyme in ATPSs can be simplified and made more efficient if a thermo-separating polymer is used instead of polyethylene glycol (PEG). The thermo separating polymer is composed of ethylene oxide-propylene oxide (EOPO) forming the top phase in the two-phase systems with salt solutions forming the bottom phase. In this two-phase system, the EOPO polymer is separated from the aqueous solution of the purified lysozyme during a thermo-separation process of the polymer, which can be repeatedly applied just as the salt solution left after the first stage of the two-phase extraction.