The production of Immunoglobulin Y (IgY) from an immunized chicken egg yolk serves numerous applications in various fields of immunology. The present work, for the first time, demonstrates the feasibility of aqueous two-phase extraction and three phase partitioning for the separation of IgY from the chicken egg yolk. The protocol deals with partitioning of IgY using polyethylene glycol 1500/potassium phosphate aqueous two-phase system and its comparison with three phase partitioning, both followed by the precipitation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried out to confirm the presence of IgY at the different steps of purification process. The IgY was preferentially partitioned to the PEG-rich top phase, especially at lower phase-volume ratio. The effect of process parameters such as tie line length and volume ratio was studied. The tie line length of 5.55% and volume ratio of 0.49 gave the best results. The yield of IgY obtained from aqueous two-phase system was 9.0mg/mL of egg yolk while that of three phase partitioning it was 6.0mg/mL.