Protonation sites in biological molecules are very important because they directly determine their structures, properties, and functions. The protonation sites and structures of the tripeptides of Gly and Ala have been investigated using infrared multiple photon dissociation (IRMPD) spectroscopy in combination with theoretical calculations. The experimental and calculated results indicate that two isomers coexist under the experimental conditions. Different protonation sites result in the different structures. If the excess proton is bound to the amino group, a cyclic structure is formed, in which the N-terminus and carbonyl oxygen of the C-terminus are linked by a strong hydrogen bond. In contrast, if the excess proton is bound to the amide carbonyl oxygen, a linear structure is formed in which a very strong hydrogen bond is formed, linking the two amide carbonyls. For the first time, IR spectroscopic evidence confirms that an amide oxygen may serve as the protonation site for peptides.