Abstract <p>(S)-2,5-Dioxypyrrolidine-1-yl-(5-(4-methylpiperazin-1-yl)-2,4-dinitrophenyl) pyrrolidine-2-carboxylate (<b>PPZ-Pro-NHS</b>) was used as the reagent for chiral derivatization to distinguish between amino acid enantiomers. PPZ-Pro-NHS, synthesized from (S)-1-(5-(4-methylpiperazin-1-yl)-2,4-dinitrophenyl) pyrrolidine-2-carboxylic acid, reacted with chiral amines under mild conditions to yield the corresponding organic derivatives. The optimal conditions for derivatization using PPZ-Pro-NHS were 25°C and 40 min in a medium containing triethylamine. A simple liquid chromatography–mass spectrometry (<b>LC</b>−<b>MS</b>) method was developed to separate chiral amino acids using reversed-phase chromatography. The amino acids were separated by isocratic or gradient elution on a Mightysil RP-18 GP column with a mobile phase containing H₂O, methanol, and 0.1% (v/v) formic acid. The enantiomers of the amino acids were fully separated with high resolution (<i>Rs</i> = 1.20–5.30) and high sensitivity (limit of detection: 85–4546 fmol) using PPZ-Pro-NHS derivatization and LC–MS analysis with both isocratic and gradient elution. The enantiomers of 16 amino acid mixtures were simultaneously separated in a single chromatographic run using a gradient mobile phase system. Thus, PPz-Pro-NHS can be used for chiral amine screening and authentication.</p>