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Extractive fermentation using aqueous biphasic systems (ABS) is a promising separation process since it provides a nondenaturing environment for biomolecules and improves the stability of cells. Due to environmental concerns and toxicity issues related with common volatile organic solvents, ionic liquids (ILs), a new class of nonvolatile alternative solvents, are being currently investigated for extraction purposes. In this work, a wide range of imidazolium-based ILs was studied aiming at obtaining new insights regarding their ability toward the formation of ABS and their capacity to the extraction of biomolecules. On the basis of the IL cations 1-ethyl-3-methylimidazolium and 1-butyl-3-methylimidazolium, the IL anion influence on ABS formation was assessed through their combination with chloride, bromide, acetate, hydrogensulfate, methanesulfonate, methylsulfate, ethylsulfate, trifluomethanesulfonate …