Several L-proline and (4R)-hydroxy-L-proline derivatives were evaluated as chiral selectors (CSs) in the separation of enantiomers by counter-current chromatography (CCC). A variety of biphasic solvent systems, all of organic/aqueous nature, were tested in order to determine the appropriate distribution for CSs and racemates (N-(3,5-dinitrobenzoyl)-(+/-)-leucine and (+/-)-ketoprofen). Successful separations of DNB-(+/-)-leucine in analogous experimental conditions allow the comparative study of the enantioselectivity displayed by the considered CSs. The low solubility of certain CSs limits their applicability for preparative purposes even for improved enantioselectivity. The effect that the nature and pH of the buffer solutions used as a component of the solvent system have on the separation was also studied.