The current study is devoted to the synthesis of amino acid ionic 1-butyl-3-methylimidazolium L-prolinate [C4MIm][L-Pro] liquid and the investigation of its possibilities as a chiral selector for the separation of amino acids (tryptophan and tyrosine) and β-blockers (carvedilol and propranolol) enantiomers. The following factors affecting the resolution of amino acids stereoisomers under ligand exchange capillary electrophoresis were established: background electrolyte pH, concentration of chelate complex, molar ratio of metal and ligand and its nature. The enantioselectivity values of 1.25 for tryptophan enantiomers and 1.17 for tyrosine enantiomers were observed when the background electrolyte consisted of 50 mM borate buffer (pH = 12.2), 20 mM [C4MIm][L-Pro], and 10 mM CuSO4. The synergetic effect of the synthesized ionic liquid and (2-hydroxypropyl)-β-cyclodextrine when added to the background electrolyte content (20 mM NaH2PO4, pH = 2.5) was discovered during the separation of carvedilol and propranolol enantiomers. The achieved resolution for carvedilol (Rs = 1.1) and propranolol (Rs = 1.6) enantiomers allowed determining the enantiomers ratio of active ingredients in drug formulations.