N-Boc/Fmoc/Z-N′-formyl-gem-diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro-inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion-trap multi-stage mass spectrometry (MSn). The MS2 collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N-Fmoc/Z-amino acids, dipeptide and tripeptide acids show the [M + Na-NH2CHO]+ ion, arising from the loss of formamide, as the base peak. Differently, the MS2 CID spectra of [M + Na]+ ion of all the N-Boc derivatives yield the abundant [M + Na-C4H8]+ and [M + Na-Boc + H]+ ions because of the loss of isobutylene and CO2 from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N-protected dipeptidyl and tripeptidyl-N′-formamides is provided by MS2 and subsequent MSn experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M-H]−, [M + HCOO]− and [M + Cl]− ions, the presence of in-source CID fragment ions deriving from the involvement of the N-protecting group. Furthermore, MSn spectra of [M + Cl]− ion of N-protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C-terminal gem-diamino residue. The present paper represents an initial attempt to study the ESI-MS behavior of these important intermediates and lays the groundwork for structural-based studies on more complex partially modified retro-inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.