Abstract: Proline derivatives have unique structures, and few reports have been published on the chiral identification of amino acids by nuclear magnetic resonance (NMR). The structures of two proline N-amide conformation isomers were analyzed in detail using ¹H NMR, ¹H-¹H correlated spectroscopy(¹H-¹H COSY), ¹H-¹H total correlation spectroscopy(¹H-¹H TOCSY), ¹H-¹H nuclear overhauser effect spectroscopy(¹H-¹H NOESY), ¹³C NMR, distortionless enhancement by polarization transfer(DEPT135°), ¹H-¹³C ¹H detected heteronuclear single quantum coherence(¹H-¹³C HSQC) and ¹H-¹³C ¹H detected heteronuclear multiple-bond correlation(¹H-¹³C HMBC) techniques, and the positions of ¹H and ¹³C NMR peaks of the derivatives were assigned. The co-existence of L- and D-type cis-trans isomers in dimethylsulfoxide (DMSO) at room temperature were determined.