A possibility of using ionic liquids based on imidazolium as modifiers of the mobile and stationary phases under the conditions of reversed-phase and hydrophilic chromatography (HILIC) for the separation of polar biologically active substances (amino acids, vitamins and drugs) has been investigated. It was shown that the introduction of imidazolium ionic liquids (IL) to the eluent led to the dynamic modification of the C18 sorbent surface which in turn led to the increased efficiency and selectivity of basic analytes separation. The realization of HILIC mode was possible due to the formation of a hydrophilic coating. Influences of the alkyl radical (C4, C6, C8, C12) length, nature of the counterion (Cl-, BF4-), IL concentration (0.1 - 20 mM), pH of the mobile phase and concentration of organic additive on the efficiency and selectivity of the separation of polar drugs had been studied. In addition, the ability of cetyltrimethylammonium bromide (CTAB) to provide HILIC conditions in the concentrations lower than critical micelle concentration (CMC) has been investigated. The possibility of determining drugs ( p- aminosalicylic acid and antibiotics levofloxacin, sparfloxacin) in blood plasma under HILIC conditions was shown.