The potential of the antibiotic vancomycin (VC) as chiral selector for the enantiomeric separation of amino acids by CE-ESI-MS/MS2 was investigated for the first time in this work. Derivatization of amino acids with FMOC-Cl was carried out to enable their interaction with VC as well as the formation of precursor ions with larger m/z which were employed in MS2 experiments. The partial filling of a coated capillary was employed to avoid the loss in MS sensitivity originated by the introduction of VC in the ionization source. Under optimized conditions, the simultaneous enantiomeric separation and unequivocal identification of 17 amino acids (two of them being nonprotein amino acids) took place in about 20 min with LODs in the micromolar range.