In the past few decades, macrocyclic antibiotic molecules have become among the most useful chiral selectors in analytical HPLC, thin-layer chromatography and capillary electrophoresis and also in preparative methods. The macrocyclic glycopeptides, such as teicoplanin, vancomycin and ristocetin A and its analogs, are perhaps the most useful selectors for the enantioseparation of nonprotected and N-protected peptides and amino acids, β-blockers, β-agonists, nonsteroidal anti-inflammatory drugs, antineoplastics and various other biologically important compounds. This article discusses the physicochemical properties, method developments, mechanisms and applications for separations on macrocyclic antibiotics. Since comprehensive reviews on the HPLC separation of biologically important analytes on macrocyclic antibiotic chiral stationary phases have been published up to the middle of the 2000s, the recent progress covers the period 2006–2010, focussing on the HPLC applications of antibiotic phases.