Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of D-amino acids typically found in biological fluids and tissues.