For the simultaneous determination of lactate (LA), 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB) and malate (MA) enantiomers, a new separation technique in a two-dimensional high-performance liquid chromatography (2D-HPLC) system following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been developed. The 2D-HPLC was composed of the reversed-phase (1D) and enantioselective (2D) separations. In the first dimension, the target hydroxy acids were separated as their D plus L mixtures on a Singularity RP18 column (1.0 mm i.d. x 250 mm), and the enantiomers were separated in the second dimension using a polysaccharide-based chiral stationary phase, Chiralpak IG (2.0 mm i.d. x 250 mm). By using the Chiralpak IG column, the LA, 2HB, 3HB and MA enantiomers were separated with resolution values of 3.47, 3.63, 6.81 and 3.72, respectively. The developed 2D-HPLC system was applied to human plasma, and trace levels of D-LA and L-3HB in addition to their major antipodes were determined. The acquired %D of LA (the percentage of D-LA over total LA) and %L of 3HB (the percentage of L-3HB over total 3HB) were 1.0 and 2.7, respectively. For 2HB, only the L-form was detected, while the MA enantiomers were not found in the human plasma.