Enantioselective dissociation in the gas phase is important for enantiomeric enrichment and chiral transmission processes in molecular clouds regarding the origin of homochirality in biomolecules. Enantioselective collision-activated dissociation (CAD) of tryptophan (Trp) and the chiral recognition ability of l-alanine peptides (l-Alan; n = 2–4) were examined using a linear ion trap mass spectrometer. CAD spectra of gas-phase heterochiral H+(d-Trp)(l-Alan) and homochiral H+(l-Trp)(l-Alan) noncovalent complexes were obtained as a function of the peptide size n. The H2O-elimination product was observed in CAD spectra of both heterochiral and homochiral complexes for n = 2 and 4, and in homochiral H+(l-Trp)(l-Ala3), indicating that the proton is attached to the l-alanine peptide, and H2O loss occurs from H+(l-Alan) in the noncovalent complexes. H2O loss did not occur in heterochiral H+(d-Trp)(l-Ala3), where NH3 loss and (H2O + CO) loss were the primary dissociation pathways. In heterochiral H+(d-Trp)(l-Ala3), the protonation site is the amino group of d-Trp, and NH3 loss and (H2O + CO) loss occur from H+(d-Trp). l-Ala peptides recognize d-Trp through protonation of the amino group for peptide size n = 3. NH3 loss and (H2O + CO) loss from H+(d-Trp) proceeds via enantioselective CAD in gas-phase heterochiral H+(d-Trp)(l-Ala3) at room temperature, whereas l-Trp dissociation was not observed in homochiral H+(l-Trp)(l-Ala3). These results suggest that enantioselective dissociation induced by chiral recognition of l-Ala peptides through protonation could play an important role in enantiomeric enrichment and chiral transmission processes of amino acids.