L-Carnosine (Car) is an endogenous dipeptide with high potential for drug discovery approaches in neurogenerative diseases, whereas TAT1 is a small arginine-rich peptide derived from the HIV-1 trans-activator protein (TAT) known to stimulate proteasome activity. Three isomeric peptides were synthetised by adding the Car moiety to the TAT1 sequence at the C- and N-termini and in the middle of the peptide sequence. High-resolution and energy-resolved CID MS/MS experiments were performed to differentiate the three isomeric peptides. At first glance, the obtained MS/MS spectra showed a high degree of similarity between the peptides. A wealth of low-intensity fragment ions peaks resulting mainly from arginine-specific neutral losses, which are useless for structural elucidation, or peaks that cannot be easily assigned were observed. Energetic study was also non-conclusive with that respect. However, Principal Component Analysis (PCA) showed that the three peptides could be clearly distinguished when the entire MS/MS spectra were considered rather than just the intensity of the precursor ions peak. Interestingly, the PCA loadings revealed the characteristic fragment ions of each peptide (although with smaller intensities) providing hints on consecutive fragmentation patterns. Some of these specific peaks could also be assigned to scrambling during fragmentation. These results demonstrate the potential of PCA as a simple chemometric tool for semi-automated peak identification in complex MS/MS spectra.