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 the large variety of their interactive sites (e.g., hydrophobic pockets, hydroxyl, amino and carboxyl groups, halogen atoms, aromatic moieties, etc.) are the reasons for their wide-ranging selectivity. The commercially available columns based on antibiotic selectors, which display complementary characteristics, are capable of separating a wide variety of enantiomeric compounds with good efficiency, good column loadability, high reproducibility, and long-term stability. These are the main reasons for the frequent use of macrocyclic antibiotic-based stationary phases in enantioselective liquid chromatography. This overview chapter provides a summary of general aspects of antibiotic-based chiral stationary phases, including their preparation and their application to direct enantioseparations of various chiral compounds, focusing on the literature published between 2018 and 2023.