Phosphoramidates of nucleosides are continuing to be an important class of rationally designed therapeutics especially as oligonucleotide analogs employed as antisense and antigene agents. A series of different nucleoside 5’-thiophosphoramidates have recently been synthesized, their electrospray ionization mass specra have been investigated. The positive and negative electrospray ionization mass spectra all gave strong pseudomolecular ions and characteristic fragment ions, however, they showed different fragmentation pathways. In the negative ion MS, the deprotonated molecule showed the base peak with loss of propylene. In the positive ion MS, the protonated molecule usually produced ions from loss of water, amino acid methyl ester, methyl alcohol, uridine and phosphoryl group. These mass spectral fragmentation pathways are helpful to determination of similarphosphoramidates of nucleosides. Phosphoramidates of nucleosides are continuing to be an important class of rationally designed therapeutics especially as oligonucleotide analogs employed as antisense and antigene agents. A series of different nucleosides 5’-thiophosphoramidates have recently been synthesized, their electrospray ionization mass specra have been investigated. The positive and negative electrospray ionization mass spectra all gave strong pseudomolecular ions and characteristic fragment ions, however, they showed different fragmentation pathways. In the negative ion MS, the deprotonated molecule showed the base peak with loss of propylene. molecules usually produced ions from loss of water, amino acid methyl ester, methyl alcohol, uridine and phosphoryl group. These mass spectral fragmentation pathways are helpful to determine of similar phosphoramidates of nucleosides.