Since their introduction by Daniel W. Armstrong in 1994, antibiotic-based chiral stationary phases have proven their applicability for the chiral resolution of various types of racemates. The unique structure of macrocyclic glycopeptides and their large variety of interactive sites (e.g., hydrophobic pockets, hydroxy, amino and carboxyl groups, halogen atoms, aromatic moieties) are the reasons for their wide-ranging selectivity. The commercially available Chirobiotic™ phases, which display complementary characteristics, are capable of separating a broad variety of enantiomeric compounds with good efficiency, good column loadability, high reproducibility, and long-term stability. These are the major reasons for the frequent use of macrocyclic antibiotic-based stationary phases in HPLC enantioseparations.This overview chapter provides a brief summary of general aspects of antibiotic-based chiral stationary phases including their preparation and their application to direct enantioseparations of various racemates focusing on the literature published since 2004.