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Macrocyclic antibiotics, especially vancomycin, represent a class of versatile chiral selectors for enantioseparations by capillary electrophoresis (CE). In this chapter, we describe the protocol for performing CE enantioseparations with vancomycin as the chiral selector. Dynamic coating of the capillary with polymers including the positively charged polyelectrolyte hexadimethrine bromide or electro-neutral poly(dimethylacrylamide) proved to be very useful in order to reduce the adsorption of vancomycin onto the capillary wall resulting in an improved separation efficiency. The partial filling technique is employed for improvement of the detection sensitivity. Utilization of these techniques makes the CE enantioseparation with vancomycin more practical and robust.