In solution, the most basic site in 4-aminobenzoic acid is the amino nitrogen, while the carboxylic acid oxygen is the most basic site in the gas phase. However, the protonation site in the gas phase has been demonstrated to depend on the ionization solvents when ionized using positive ion mode electrospray ionization (ESI). In many of these studies, collision-activated dissociation (CAD) was used to differentiate the protomers. To explore the influence of different CAD conditions on the manifested protonation site, 4-aminobenzoic acid dissolved either in 1:1 acetonitrile−water or 3:1 methanol−water was ionized by ESI and subjected to three different CAD experiments in a linear quadrupole ion trap/orbitrap mass spectrometer. Based on in-source CAD (ISCAD) and beam-type medium-energy CAD (MCAD), the proton resided on the nitrogen atom (N-protomer) when acetonitrile−water was used as the solvent system but on the oxygen atom (O-protomer) when methanol−water was used. Interestingly, a predominant Nprotomer was observed when CAD was performed in the linear quadrupole ion trap (ITCAD), irrespective of the solvents used, in disagreement with literature. This unexpected result is rationalized based on the formation of long-lived water clusters of varying sizes for the protomers in the quadrupole ion trap due to residual water, low ion kinetic energies, long ion storage times, and relatively high pressure. Further, addition of extra water into the quadrupole ion trap resulted in nearly identical protomer distributions for both protomers. Therefore, this distribution must be near the equilibrium distribution caused by the presence of water clusters of varying sizes, some favoring the N-protomer and others the O-protomer.