Abstract: Ionic liquids (ILs) are widely used in separation methods such as chromatography and capillary electrophoresis due to their unique properties. This review discusses the prospects for using imidazole-based ILs as electrophoretic system modifiers in capillary zone electrophoresis and micellar and microemulsion electrokinetic chromatography for the determination of biologically active substances (amino acids, catecholamines, steroid hormones, and polyphenolic antioxidants). New approaches to selective electrophoretic determination and efficient online preconcentration of analytes with the participation of imidazolium ILs are considered; the application of developed approaches to the analysis of natural objects (urine, blood plasma, and plant extracts) is exemplified.