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Metabolomics profiling using liquid chromatography-mass spectrometry (LC-MS) has become an important tool in biomedical research. However, resolving enantiomers still represents a significant challenge in the metabolomics study of complex samples. Here, we introduced <i>N,N</i>-dimethyl-<span style="font-variant: small-caps;">l</span>-cysteine (dimethylcysteine, DiCys), a chiral thiol, for the <i>o</i>-phthalaldehyde (OPA) derivatization of enantiomeric amine metabolites. We took interest in DiCys because of its potential for multiplex isotope-tagged quantification. Here, we characterized the usefulness of DiCys in reversed-phase LC-MS analyses of chiral metabolites, compared against five commonly used chiral thiols: <i>N</i>-acetyl-<span style="font-variant: small-caps;">l</span>-cysteine (NAC); <i>N</i>-acetyl-<span style="font-variant: small-caps;">d</span>-penicillamine (NAP); isobutyryl-<span style="font-variant: small-caps;">l</span>-cysteine (IBLC); <i>N</i>-(<i>tert</i>-butoxycarbonyl)-<span style="font-variant: small-caps;">l</span>-cysteine methyl ester (NBC); and <i>N</i>-(<i>tert</i>-butylthiocarbamoyl)-<span style="font-variant: small-caps;">l</span>-cysteine ethyl ester (BTCC). DiCys and IBLC showed the best overall performance in terms of chiral separation, fluorescence intensity, and ionization efficiency. For chiral separation of amino acids, DiCys/OPA also outperformed Marfey’s reagents: 1-fluoro-2-4-dinitrophenyl-5-<span style="font-variant: small-caps;">l</span>-valine amide (FDVA) and 1-fluoro-2-4-dinitrophenyl-5-<span style="font-variant: small-caps;">l</span>-alanine amide (FDAA). As proof of principle, we compared DiCys and IBLC for detecting chiral metabolites in aqueous extracts of rice. By LC–MS analyses, both methods detected twenty proteinogenic <span style="font-variant: small-caps;">l</span>-amino acids and seven <span style="font-variant: small-caps;">d</span>-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val), but DiCys showed better analyte separation. We conclude that DiCys/OPA is an excellent amine-derivatization method for enantiomeric metabolite detection in LC-MS analyses.