The positive and negative ESI-MS/MS spectra of N-ethoxy(phenyl) phosphoryl amino acids(EPP-AA) were investigated by electrospray ionization(ESI) ion trap mass spectrometry. The fragmentation pathways of [M+Na] + and [M-H] - ions are proposed and rationalized. The observation may have some potential applications in the interpretation of the MS/MS spectra of novel N-phosphoryl compounds. The complexity of MS/MS spectra of EPP-AA [M+Na] + ions is decreased compared with that of N-dialkyloxyphosphoryl amino acid. Therefore, the new phos- phonamidate method may be considered one of the superior methods that can be used in sequencing peptides and proteins extensively. The positive and negative ESI-MS / MS spectra of N-ethoxy (phenyl) phosphoryl amino acids (EPP-AA) were investigated by electrospray ionization (ESI) ion trap mass spectrometry. The fragmentation pathways of [M + Na] + and [ MH] - ions are proposed and rationalized. The observation may have some potential applications in the interpretation of the MS / MS spectra of novel N-phosphoryl compounds. The complexity of MS / MS spectra of EPP-AA [M + Na] + ions is decreased compared with that of N-dialkyloxyphosphoryl amino acid. Thus, the new phos- phonamidate method may be considered one of the superior methods that can be used in sequencing peptides and proteins extensively.