We apply cryogenic ion vibrational predissociation (CIVP) spectroscopy to characterize the O 2 attachment motif in a prototypical peroxo manganese (III) reaction intermediate. In this approach, species are extracted from solution using electrospray ionization and cooled in a 30K ion trap. The infrared spectrum is then obtained by monitoring the photoinduced evaporation of a single, weakly bound N 2 molecule as a function of laser wavelength. Because the resulting CIVP action spectrum is linear in laser fluence, the pattern of well-resolved transitions can be directly compared with harmonic spectra calculated for predicted local minima using density functional theory (DFT). The assignment of the OO stretching band derived from the activated O 2 ligand is established by following the evolution of the bands with 18 O 2 substitution, and the energy of this transition indicates that O 2 is bound side-on to the Mn center. The successful application of CIVP to this class of compounds opens the way for sensitive spectroscopic characterization of weakly abundant species in complex solution environments.