The effect of phase forming components, pH, neutral salt (NaCl) addition on partitioning of bovine serum albumin (BSA) in PEG2000-sodium citrate based aqueous two-phase system was investigated at 25, 35, and 45C. The systems were prepared by varying the phase forming components concentration at a pH of 5.0, 6.0, 7.0, 8.0, and 9.0 with different NaCl salt concentrations of 0.05M, 0.1M, 1.5M, 0.2M, 0.3M, and 0.4M. The affinity of the BSA for the lower phase increases with increase in pH, due to increase in ratio of trivalent to divalent citrate ions in the two-phase system. It was confirmed that the partition of BSA depends on the relative hydrophobicity of the compounds as well as their charge. Optimum level of pH and NaCl concentration for the partitioning of bovine serum albumin was determined.