A new pre-column derivatization reagent with a 6-methoxy-4-quinolone (6-MOQ) moiety for amino acid analysis, 2,5-dioxopyrrolidin-1-yl(2-(6-methoxy-4-oxoquinolin-1(4H)-yl)ethyl) carbonate (6-MOQ-EtOCOOSu), was designed and synthesized. 6-MOQ is a thermo/photostable fluorophore with a high proton-affinity site and sensitive determination could be carried out by a fluorescence detector and also by an electrospray ionization mass spectrometer. Derivatization of amino acids with 6-MOQ-EtOCOOSu was completed within 1 min under mild basic conditions at room temperature. The 6-MOQ derivatives of all chiral proteinogenic amino acids were separated using the combination of three enantioselective columns, Chiralpak QN–AX, Chiralpak ZXIX(+), and KSAACSP-001S, with separation factors of higher than 1.07. The present reagent enables the sensitive determination of amino acid enantiomers, and the values of LLOD using a chiral-HPLC–MS/MS system were 0.05–50 fmol/injection.