Absolute bond dissociation energies (BDEs) of glycylglycine (GG) and glycylglycylglycine (GGG) to sodium and-potassium cations and sequential bond energies of glycine (G) in Na(+)G(2) were determined experimentally by threshold collision-induced dissociation (TCID) in a guided ion beam tandem mass spectrometer. Experimental results showed that the binding energies follow the order of Na+ > K+ and M(+)GGG > M(+)GG > M(+)G. Theoretical calculations at the B3LYP/6-311 +G(d) level show that all complexes had charge-solvated structures (nonzwitterionic) with either [CO,CO] bidentate or [N,CO,CO] tridentate coordination for M+GG complexes, [CO,CO,CO] tridentate or [N,CO,CO,CO] tetradentate coordination for M+GGG complexes, and [N,CO,N,CO] tetradentate coordination for Na(+)G(2). Ab initio calculations at three different levels of theory (B3LYP, B3P86, and MP2(full) using the 6-311 +G(2d,2p) basis set with geometries and zero-point energies calculated at the B3LYP/6-311 +G(d) level) show good agreement with the experimental bond energies. This study demonstrates for the first time that TOD measurements of absolute BDEs can be successfully extended to biological molecules as complex as a tripeptide.