To separate lactoferrin from whey, extraction in three-phase systems was applied (known as a threephase partitioning ‘TPP’ method). In the research study, 72 variants of three-phase systems were investigated; they differed in the degree of saturation of whey with ammonium sulphate, pH, and whey to tertbutanol volume ratio. In the phases formed, the concentration of lactoferrin and total protein were measured in order to determine the separation parameters such as purification factor and yield of recovered lactoferrin. Based on the separation parameters, obtained through the experiment, a mathematical model was constructed. The optimal composition of the three-phase system, determined on the basis of this model, consisted of the whey having a pH value of 10, saturated with ammonium sulphate at a concentration of 35 %, whereas the whey to tert-butanol volume ratio was 1 : 0.5. Those conditions made it possible to obtain a lactoferrin preparation in the bottom phase of the three-phase system, and the purification factor thereof was ca. 3. The extraction yield of lactoferrin in this phase was 26 %.