Direct and indirect high-performance liquid chromatographic methods were developed for the enantioseparation of beta-amino acids (beta-substituted-beta-alanines). Direct separation involved the application of chiral columns: Crownpak CR(+), Chirobiotic T and Chirobiotic R. Indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate or N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alanineamide (Marfey's reagent), with subsequent separation on an achiral column. The chromatographic conditions were varied to achieve optimum separation.