A group of nearly forty-eight (48) of surfactants based on ionic liquids (ILs) has been described. The physicochemical and interfacial behavior for several of these IL-based surfactants has been reported since 2004. This review attempts to summarize the important applications that IL-based surfactants have had in analytical chemistry and separation science. Thus, they have been successfully used as extractant systems as substitutes to conventional organic solvents in extraction schemes, or by modifying the chemical structure of conventional extractant sorbents. The analytical performance of these novel IL-based surfactants has been shown to be better than conventional solvents and even better than conventional cationic surfactants. IL-based surfactants have also been used as mobile phase and stationary phase modifiers in high-performance liquid chromatography (HPLC), as modifiers of the electrokinetic chromatographic (EKC) medium, or as the micellar component in capillary electrophoresis (CE). Thus, IL-based surfactant methods constitute a promising and developing area within separation science.