Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which provides unique selectivities compared to reversed-phase chromatography (RPLC) for peptide separations. Separations by HILIC/CEX are effected by linear increasing salt gradients in the presence of acetonitrile (up to 90%), which promotes hydrophilic interactions overlayed on ionic interactions with the ion-exchange matrix. In the present study, the utility of HILIC/CEX has been extended to the separation of cyclic peptides in the form of synthetic model analogues of gramicidin S: Series 1 comprised six 10-residue cyclic peptide analogues which exhibited amphipathic, rigid β-pleated sheet conformation; Series 2 comprised 14-residue cyclic diastereomeric analogues of gramicidin S, where only the enantiomeric configuration of a single amino acid side-chain is varied from peptide to peptide. Observation of the retention behaviour of these two series of cyclic peptides during HILIC/CEX and RPLC confirmed not only the excellent complementarity of these two chromatographic modes but also highlighted the dramatic separations achievable by the mixed-mode approach.