Nucleotide phosphoramidates are prodrugs which effectively deliver the active nucleotide to target tissues. It was shown that the individual phosphoramidate diastereomers have different antiviral activity, although the active nucleotide is the same. Therefore, a fast and simple analytical method is needed to characterize the individual diastereomeric phosphoramidate prodrugs. Stock solutions of diastereomeric nucleotide phosphoramidate prodrugs, i.e., 5'-phosphate derivatives of the β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methyluridine nucleotide, were made in 25% acetonitrile to achieve a final concentration of 10 µg/mL. The samples were studied using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The MS/MS spectra of diastereomeric pairs showed substantial differences in the relative abundances of a characteristic ion in negative mode, which is proposed to be a cyclic phosphoramidate ion. Results were confirmed by the MS/MS spectrum of an analog without the NH proton and deuterium exchange experiment. Furthermore, the diastereomer-specific fragmentation behavior in negative ESI-MS was used to characterize a series of nucleotide phosphoramidates with different amino acid and aromatic substituents. An HPLC/MS/MS method was developed for the differentiation of the diastereomers of phosphoramidate prodrugs. In negative mode MS/MS spectra, the cyclic phosphoramidate ions yielded unambiguous distinction. This method presented a rapid and simple way for the characterization of nucleotide phosphoramidates.