During the past 13 years, ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been the focus of remarkable interest and research. They have shown to be promising separation strategies for the most diverse compounds, resulting mainly from their tailoring ability offered by the current large number of IL chemical structures. A significant number of scientific manuscripts on IL-based ABS have been reported up to date, either on their characterization by attempting the determination of their phase diagrams or by exploring their viability on the separation of target compounds. The molecular-based scenario which rules the phase demixing in these systems and the comprehension of the best conditions and systems for improved separation performance have been ascertained. Both IL and other phase-forming components chemical structures, as well as pH and temperature effects, have been deeply evaluated in order to infer on their liquid–liquid demixing aptitude. On the other hand, possible and promising applications of IL-based ABS have been disclosed by investigating their role on the extraction of a wide plethora of biomolecules and compounds, e.g. amino acids, proteins, alkaloids, phenolic acids and antibiotics, amongst others. In fact, IL-based ABS proved to be outstanding separation platforms compared to more traditional polymer-based systems due to their wider hydrophilic–hydrophilic range which allows enhanced and selective extractions. Concentration factors up to 1000-fold and purification factors up to 245 have been reported with IL-based ABS. In this chapter we review and summarize the definition of IL-based ABS; describe the main phase-forming components used for their creation; define the fundamentals behind the formation of two aqueous-rich phases; demonstrate and show examples of their applications at the extraction, purification and concentration levels; evaluate the most promising applications of IL-based ABS; and discuss their future applicability.