The number of degrees of freedom-dependent stability of ions and ion–neutral non-covalent complexes under collision-induced dissociation (CID) conditions was studied in a quadrupole ion trap mass spectrometer. It was found that the stability of ions as probed by energy-variable CID has a linear dependence on the total number of degrees of freedom for the ions (or ion–neutral complexes) with the same (or nearly the same) bonding energy. The slope of such a stability vs number of degrees of freedom dependence correlates with the binding energy. Proton-bound amine dimers display the lowest slope as they have weak bonds. Breaking covalent bonds will result in much greater slopes. In addition to the binding energy, the vibrational frequencies of the ion also affect the stability vs number of degrees of freedom behavior. Studying such a dependence of the CID stability in a system paves the way for direct relative binding energy comparisons. The application of this approach is demonstrated by testing the relative heme affinities of anti-malaria drugs and related compounds. Copyright © 2004 John Wiley & Sons, Ltd.