2-hydroxyglutarate (2HG) is a potent competitor of α-ketoglutarate (α-KG) and can inhibit multiple α-KG dependent dioxygenases that function on the epigenetic modifications. The accumulation of 2HG contributes to elevated risk of malignant tumors. 2HG carries an asymmetric carbon atom in its carbon backbone and differentiation between D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG) is crucially important for accurate diagnosis of 2HG related diseases. Here we developed a strategy by chiral derivatization combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive determination of D-2HG and L-2HG enantiomers. N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was used to derivatize 2HG. The formed diastereomers by TSPC labeling can efficiently improve the chromatographic separation of D-2HG and L-2HG. And derivatization by TSPC could also markedly increase the detection sensitivities by 291 and 346 folds for D-2HG and L-2HG, respectively. Using the developed method, we measured the contents of D-2HG and L-2HG in clear cell renal cell carcinoma (ccRCC) tissues. We observed 12.9 and 29.8 folds increase of D-2HG and L-2HG, respectively, in human ccRCC tissues compared to adjacent normal tissues. The developed chiral derivatization combined with LC-ESI-MS/MS analysis offers sensitive determination of D-2HG and L-2HG enantiomers, which benefits the precise diagnosis of 2HG related metabolic diseases.