The fragmentation reactions of isomeric dipeptides containing alpha- and beta-alanine residues (alphaAla-alphaAla, alphaAla-betaAla, betaAla-alphaAla, and betaAla-betaAla) were studied using a combination of low-energy and energy resolved collision induced dissociation (CID). Each dipeptide gave a series of different fragment ions, allowing for differentiation. For example, peptides containing an N-terminal beta-Ala residue yield a diagnostic imine loss, while lactam ions at m/z 72 are unique to peptides containing beta-Ala residues. In addition, MS(3) experiments were performed. Structure-specific fragmentation reactions were observed for y(1) ions, which help identify the C-terminal residue. The MS(3) spectra of the b(2) ions are different suggesting they are unique for each peptide. Density functional theory (DFT) calculations predict that b(2) ions formed via a neighboring group attack by the amide are thermodynamically favored over those formed via neighboring group attack by the N-terminal amine. Finally, to gain further insight into the unique fragmentation chemistry of the peptides containing an N-terminal beta-alanine residue, the fragmentation reactions of protonated beta-Ala-NHMe were examined using a combination of experiment and DFT calculations. The relative transition-state energies involved in the four competing losses (NH(3), H(2)O, CH(3)NH(2), and CH(2)=NH) closely follow the relative abundances of these as determined via CID experiments.