Aqueous biphasic systems (ABS) have positive aspects associated with the low cost, eco-friendly system, biomolecule separation ability, and high extraction yield. The presence of quaternary ammonium salts on the ABS, e.g., cholinium chloride ([N-111(2OH)]Cl), confers to these systems unusual characteristics for biomolecule separation and purification. In this work, PEG-based ABS employing cholinium chloride combined with inorganic and organic salts, to obtain ions at equivalent concentrations, were evaluated. In general, it was observed that the presence of cholinium chloride decreases the biphasic region compared to the ternary system. For all systems, the top phase is richer in polymer, and the bottom phase is richer in salt, regardless of the cholinium chloride presence. Furthermore, the influence of cholinium chloride through the partition coefficient of different kinds of biomolecules (alkaloids, amino acids, and phenolic compounds) was estimated. The cholinium chloride presence demonstrated to be able to tune the partition behavior due to its affinity with the evaluated biomolecules.