A new class of diastereomeric pairs of non-natural amino acid peptides derived from butyloxycarbonyl (Boc-)protected cis-(2S,3R)- and trans-(2S,3S)-β-norbornene amino acids including a monomeric pair have been investigated by electrospray ionization (ESI) tandem mass spectrometry using quadrupole time-of-flight (Q-TOF) and ion-trap mass spectrometers. The protonated cis-BocN-β-nbaa (2S,3R) (1) (βnbaa = β-norbornene amino acid) eliminates the Boc group to form [M+H–Boc+H]+, whereas an additional ion [M+H–C4H8]+ is formed from trans-BocN-β-nbaa (2S,3S) (2). Similarly, it is observed that the peptide diastereomers (di-, tri- and tetra-), with cis-BocN-β-nbaa (2S,3R)- at the N-terminus, initially eliminate the Boc group to form [M+H–Boc+H]+ which undergo further fragmentation to give a set of product ions that are different for the peptides with trans-BocN-β-nbaa (2S,3S)- at the N-terminus. Thus the Boc group fragments differently depending on the configuration of the amino acid present at the N-terminus. It is also observed that the peptide bond cleavage in these peptides is less favoured and most of the product ions are formed due to retro-Diels-Alder fragmentation. Interestingly, sodium-cationized peptide diastereomers mainly yield a series of retro-Diels-Alder fragment ions which are different for each diastereomer as they are formed starting from [M+Na–Boc+H]+ in peptides with cis-BocN-β-nbaa (2S,3R)- at the N-terminus, and [M+Na–C4H8]+ in peptides with trans-BocN-β-nbaa (2S,3S)- at the N-terminus. All these results clearly indicate that these diastereomeric pairs of peptides yield characteristic product ions which help distinguish the isomers. Copyright © 2009 John Wiley & Sons, Ltd.