This review summarizes the chiral high performance liquid chromatographic (HPLC) direct methods with different class of macrocyclic antibiotics as chiral selectors. In the past few decades, these organic macromolecules have become one of the most useful chiral selectors in analytical HPLC, thin layer chromatography (TLC) and capillary electrophoresis (CE) methods and also in preparative methods. The macrocyclic glicopeptides, such as teicoplanin, vancomycin and ristocetin, seem to be the most useful selectors for the enantioseparation of compounds with primer amino groups. Especially in wild range of amino acids and proteins. In this work the physicochemical properties, the efficiency of these antibiotics and the mechanism of separation were discussed in reversed phase (RPM), polar organic and ionic mode (POM, PIM) and in normal phase system (NPM).