The chiral separations of small peptides is an important challenge in the biological and medical sciences, because different stereoisomers of chiral drugs can often possess different pharmacological, pharmacokinetic, and/or toxicological activities. Commercially available crown ether chiral stationary phases based on S-(3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 (CROWNPAK CR-I (+)) and (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (ChiroSil RCA (+)) have been successfully used for separating enantiomers of various racemic compounds containing primary amino groups. In this investigation, enantioresolution of more complex model analyte - tetrapeptide Tyr-Arg-Phe-Lys-NH2, has been reported on crown ether chiral stationary phases. Organic and acidic modifier content in aqueous mobile phase was tested. All Tyr-Arg-Phe-Lys-NH2 stereoisomers showed U-shaped retention plots, based on ACN content in mobile phase. Increased retention of tetrapeptide stereoisomers was observed at low (<35%) and at high (>70%) acetonitrile content in the mobile phase, indicating that different separation mechanisms are most likely involved. As a result, baseline separation of all eight tetrapeptide enantiomer pairs was achieved under isocratic elution mode on both chiral columns.