1-Allyl-3-methylimidazolium chloride [Amim]­[Cl] and 1-butyl-3-methylimidazolium chloride [Bmim]­[Cl] are water-soluble ionic liquids (ILs) that can from an aqueous two-phase system (ATPS) when mixed with specific salts. Herein, we prepared [Amim]­[Cl]- and [Bmim]­[Cl]-ATPSs by adding the salts (K2CO3, K2HPO4). To investigate the phase separation behavior of the IL-ATPSs, binodal curves were drawn at different temperatures and the length and slope of the tie lines were analyzed. The [Bmim]­[Cl]/K2HPO4 system underwent two-phase separation at lower temperature conditions, suggesting that the phase separation might depend on the salting-out effect in the bottom phase. Using the IL-ATPS, the distribution coefficients, K aa, of amino acids were determined and used to characterize the hydrophobicity index (HF) between the top and bottom phases, which is a good indicator to understand the molecular partitioning behaviors in conventional ATPSs. The HF values of the IL-ATPSs were in the range 0.13–0.41 mol/kJ; these values were almost the same as the HF values reported for an ATPS composed of poly­(ethylene glycol) and salt.