The fragmentation pathways of protonated H-Gly-Gly-Sar-OH and H-Gly-Sar-Sar-OH are investigated by using both computational and experimental techniques. The main goal of these studies is to further investigate which factors determine the branching ratio of the b2-y1 (Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2002, 16, 375.) and "diketopiperazine" (Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Anal. Chem. 1993, 65, 1594.) pathways of protonated tripeptides. Protonated H-Gly-Sar-Sar-OH represents a sensitive test for the branching ratio of the b2-y1 and "diketopiperazine" pathways since this ion cannot produce y1 ions on the b(-y1 channel but only b2 ions. Protonated H-Gly-Gly-Sar-OH and H-Gly-Sar-Sar-OH exhibit very different fragmentation behavior under the investigated experimental conditions. The former fragments forming mainly y1 ions (maximum abundance of the b2 and y2 ions is approximately 15%), while the latter produces mainly b2 ions while at larger internal energies the a2, y2, and y1 ions become also moderately abundant. Theoretical modeling and analysis of the main fragmentation pathways indicate that the majority of the b2 and y1 ions of protonated H-Gly-Gly-Sar-OH and the b2 ions of H-Gly-Sar-Sar-OH are formed on the b2-y1 pathway.