Doubly deprotonated adenosine 5′-diphosphate ([ADP–2H]2−) and adenosine 5′-triphosphate ([ATP–2H]2−) dianions were investigated using infrared multiple photon dissociation (IR-MPD) and photoelectron spectroscopy. Vibrational spectra acquired in the X–H stretch region (X = C, N, O) and augmented by isotope-labelling were compared to density functional theory (DFT) calculations at the B3LYP/TZVPP level. This suggests that in [ATP–2H]2− the two phosphate groups adjacent to the ribose ring are preferentially deprotonated. Photoelectron spectra recorded at 4.66 and 6.42 eV photon energies revealed adiabatic detachment energies of 1.35 eV for [ADP–2H]2− and 3.35 eV for [ATP–2H]2−. Repulsive Coulomb barriers were estimated at ∼2.2 eV for [ADP–2H]2− and ∼1.9 eV for [ATP–2H]2−. Time-dependent DFT calculations have been used to simulate the photoelectron spectra. Photodetachment occurs primarily from lone pair orbitals on oxygen atoms within the phosphate chain.