Partitioning of methylene blue (MB) was studied in aqueous two phase systems (ATPS) prepared by mixing aqueous solutions of polymers or of a triblock copolymer with aqueous salt solutions. The partition coefficient (K) of MB in ATPS was investigated as a function of tie line length (TLL), electrolyte nature (Na2SO4 and Li2SO4), polymer molar mass, hydrophobicity of the polymers, and temperature. MB molecules concentrated preferentially in the top phase (the polymer rich phase). The partitioning behavior of MB was dependent on tie line length and polymer molar mass. It was observed that the MB partition coefficient is dependent on electrolyte nature, following the relative order Na2SO4 > Li2SO4. Values of K for MB decreased with increases in the hydrophobicity of the top phase. Higher temperatures facilitated the transfer of MB molecules to the top phase, indicating an endothermic process.