Chiral amino acid metabolomics is a novel omics approach recognizing D- and L-amino acids independently. Because several D-amino acids besides large amounts of L-amino acids have been found in mammalian tissues and physiological fluids as intrinsic physiologically active substances, analysis of chiral amino acids is expected for the diagnosis of various diseases. For the sensitive analysis, amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and determined by a two-dimensional HPLC system. In the first dimension of the 2D-HPLC system, NBD-amino acids were separated by a microbore-monolithic ODS column, and the target fractions were on-line collected to the multi-loop device. These fractions were successively transferred to the enantioselective column representing the second dimension, and the D- and L-amino acids were determined. For the separation of all amino acid enantiomers a KSAACSP-001S column (1.5 x 250 mm, originally designed) was used, and for the hydrophobic amino acids a Chiralpak QN-AX column (1.5 x 150 mm, CTE) was used. Detection of these NBD-amino acids was carried out by the fluorescence detectors and also by an ESI-tandem mass spectrometer. The present 2D-HPLC system was applied to the diagnosis of metabolic disorders of amino acids including phenylketonuria (PKU) and maple syrup urine disease (MSUD). A mouse strain deficient in phenylalanine hydroxylase was used for the model animal of PKU, and a mouse strain deficient in branched-chain alpha-keto acid dehydrogenase complex was used for MSUD. In the plasma and urine of PKU model mice, higher amounts of D-Phe and L-Phe were observed compared with those in the control mice. In the plasma and urine of MSUD model mice, higher amounts of branched aliphatic amino acid enantiomers were observed compared with those in the control mice. Especially, D-Ile and L-allo-Ile are the effective biomarkers and further studies focusing on the early diagnosis are in progress.