A combination of electrospray ionisation (ESI), multistage and high-resolution mass spectrometry experiments is used to examine the gas-phase fragmentation reactions of the three isomeric phenylalanine derivatives, α-phenylalanine, β2-phenylalanine and β3-phenylalanine. Under collision-induced dissociation (CID) conditions, each of the protonated phenylalanine isomers fragmented differently, allowing for differentiation. For example, protonated β3-phenylalanine fragments almost exclusively via the loss of NH3, only β2-phenylalanine via the loss of H2O, while α- and β2-phenylalanine fragment mainly via the combined losses of H2O + CO. Density functional theory (DFT) calculations were performed to examine the competition between NH3 loss and the combined losses of H2O and CO for each of the protonated phenylalanine isomers. Three potential NH3 loss pathways were studied: (i) an aryl-assisted neighbouring group; (ii) 1,2 hydride migration; and (iii) neighbouring group participation by the carboxyl group. Finally, we have shown that isomer differentiation is also possible when CID is performed on the protonated methyl ester and methyl amide derivatives of α-, β2- and β3-phenylalanines. Copyright © 2010 John Wiley & Sons, Ltd.