The potential use of ionic liquids (ILs) as adjuvants in typical polymer-salt aqueous systems for the separation and purification of vital biomolecules is investigated. An innovative study involving the addition of various imidazolium-based ILs to conventional PEG/inorganic salt aqueous biphasic systems (ABS), aiming at controlling their phase behaviour and extraction capability for L-tryptophan, is carried out here. For this purpose, phase diagrams and respective tie-lines for PEG 600/Na₂SO₄ ABS with the addition of small quantities of IL were established. In addition, the partition coefficients of L-tryptophan were determined in those systems. The results obtained indicate that the addition of small amounts of IL to the typical PEG/inorganic salt aqueous systems could largely control the extraction efficiency for L-tryptophan, and that efficiency depends on the IL employed. Salting-in inducing ILs enhance the partition coefficient of L-tryptophan for the PEG-rich phase while salting-out inducing ILs decrease the partitioning of the amino acid. These results are an interesting advance in biotechnological separation processes regarding the extraction of biomolecules that could be used instead of the common approach of PEG functionalization.