The intrinsic ionization energy of a base in DNA plays a critical role in determining the energies at which damage mechanisms may emerge. Here, a two-photon resonance-enhanced ionization scheme is presented that utilizes the 1ππ* transition, localized on the DNA base, to elucidate the base-specific ionization in a deprotonated nucleotide. In contrast to previous reports, the scheme is insensitive to competing ionization channels arising from the sugar–phosphate backbone. Using this approach, we demonstrate that for all bases except guanine, the lowest electron detachment energy corresponds to detachment from the sugar–phosphate backbone and allows us to determine the lowest adiabatic ionization energy for the other three bases for the first time in an isolated nucleotide.