The separation of enantiomers by liquid chromatography and capillary electrophoresis is important in pharmaceutical, environmental, and agricultural analysis. Of the different types of chiral selector used for enantiomer resolution the macrocyclic antibiotics are considered particularly effective. Only eight antibiotics, vancomycin, teicoplanin, thiostrepton, rifamycin B, kanamycin, streptomycin, fradiomycin, and ristocetin A have yet been used as chiral selectors for separation of the enantiomers of a variely of compounds by liquid chromatography and capillary electrophoresis. This review describes the chemistry of these antibiotics, the effect of chromatographic conditions on enantioselectivity, the mechanism of resolution, the applications and limitations of the compounds, and detection in liquid chromatography and capillary electrophoresis with these antibiotics as the chiral selectors.