The neutral species generated during the collisionally activated dissociation (CAD) of the protonated dipeptides Ala–Leu, Ala–Ile, Leu–Ala and Ile–Ala are post-ionized by collision and detected in neutral fragment-reionization (+NfR+) mass spectra. These spectra are dominated by fragment ions diagnostic for the C-terminal amino acid, as confirmed by comparison with reference collision-induced dissociative ionization (CIDI) mass spectra of authentic Ala, Leu and Ile. CIDI of Leu and Ile yields distinct and structurally indicative products. These are also observed upon +NfR+ of [Ala–Leu]H+ and [Ala–Ile]H+, respectively, thus allowing one to deduce the correct sequence. In contrast, [Leu–Ala]H+ and [Ile–Ala]H+, which possess identical C-termini, form no differentiating neutral losses upon CAD.