Four chiral derivatizing reagents (CDR 1-4), namely, FDNP-L-Ala, FDNP-L-Val, FDNP-L-Phe, and FDNP-L-Leu, were synthesized using microwave (MW) irradiation by substituting one of the fluorine atoms in difluoro dinitro benzene (DFDNB) with L-Ala, L-Val, L-Phe, and L-Leu (CDR 1-4). The other set of CDRs, namely, FDNP-L-Phe-NH2, FDNP-L-Val-NH2, and FDNP-L-Leu-NH2, was also prepared. These reagents were used for synthesis of diastereomers of 18 proteinogenic and 08 non-proteinogenic amino acids, which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixture of aq.TFA and acetonitrile with UV detection at 340 nm. The reagents were used for resolution of a complex mixture of 18 racemic proteinogenic amino acids in a single chromatographic run of 65 min and to determine concentration of the D: -amino acid in a solution of DL: -amino acid. The resolution (RS) and selectivity (alpha) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for accuracy, precision, limit of detection (LOD), and limit of quantification. LOD is 0.001% impurity of D-enantiomer.