The fragmentation patterns of N-diisopropyloxyphosphoryl-l-α-Ala (DIPP-l-α-Ala), N-diisopropyloxyphosphoryl-d-α-Ala (DIPP-d-α-Ala), N-diisopropyloxyphosphoryl-β-Ala (DIPP-β-Ala) and N-diisopropyloxyphosphoryl-γ-amino butyric acid (DIPP-γ-Aba) were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). DIPP-d-α-Ala showed the same fragmentation pathways as DIPP-l-α-Ala. In the fragmentation of protonated DIPP-β-Ala, the characteristic fragment ion [M+H−2C 3 H 6 −H 2 O−CH 2 CO] + appeared and could be used to distinguish β-Ala from l-α-Ala and d-α-Ala through tandem mass spectra, even though they possess the same molecular weight. In the fragmentation of protonated DIPP-γ-Aba, the break of PN bond occurred and an interesting protonated lactam ion with five-membered ring was generated. Furthermore, in the MS 3 spectrum of [M+Na−2C 3 H 6 ] + ion of DIPP-γ-Aba, a strong intensity of unique fragment ion, namely lactam-sodium adduct with five-membered ring, was observed, which could be considered as a mark for γ-amino acids. The stepwise fragmentations of their [M+Na] + ions and [M−H] − ions showed that they all underwent a PN to PO bond migration through a five-membered or six-membered or even seven-membered ring transition state, respectively, which supported the great affinity of hydroxyl for phosphoryl group.