A multiple heartcut (MHC) 2D-UHPLC method with UV detection has been developed for the enantioselective analysis of complex amino acid mixtures in a single run. The MHC method is based on an achiral gradient RPLC separation with 1.8 μm C18 phase (100 × 2.1 mm ID column) in the first dimension (D) and enantioselective isocratic separation on a tert-butylcarbamoylquinine-based 2.7 μm Coreshell particle column (50 × 3 mm ID) in the second dimension (D). Pre-column derivatization has been performed with Sanger's reagent (2,4-dinitrofluorobenzene) yielding chromogenic 2,4-dinitrophenylated amino acids (DNP-AAs). Heartcuts of 40 μL fractions of the D peaks were sampled into the D system via a two-position four-port dual valve connected to two loop decks each equipped with six 40 μL parking loops. Using this setup, 25 amino acids (20 proteinogenic plus allo-Thr, allo-Ile, homoserine (Hse), Orn, β-Ala) have been analyzed enantioselectively in a fully automated manner with a single chiral column within 130 min total run time (D and D). All D separations together took 101.5 min (29 cuts with 3.5 min run time each) and thus the total analysis time was quite efficiently utilized. Faster separations were restricted by some software constraints which did not allow to adjust run times in D individually. The practical utility of this enantioselective MHC method is documented by application for the absolute configuration determination of the amino acids in gramicidin and bacitracin. Further optimizations should lead to a generic enantioselective amino acid analyzer for the quality control of synthetic peptides and the structural characterization of non-ribosomal peptides.