The rupture forces of individual ß-cyclodextrin (ß-CD)-ferrocene host-guest complexes in an aqueous medium were measured by dynamic single molecule force spectroscopy using an atomic force microscope (AFM). The thiol-derivatized ferrocene guest was immobilized in self-assembled monolayers on gold-coated AFM tips, while the heptasulfide ß-CD host was self-assembled onto atomically flat Au(111) substrates. The effects of the alkyl spacer length of the ferrocene adsorbates, the relative concentration of ferrocene in the mixed monolayer on the AFM tip, and the unloading rate on the observed molecular unbinding events were studied. Depending on the concentration of ferrocene moieties on the AFM tip, multiple or predominantly single pull-off events were observed. A statistical analysis showed that the observed rupture forces are integer multiples of one fundamental force quantum of 55 ± 10 pN, which is attributed to the rupture of a single host-guest complex. This force quantum is found to be independent of the number of interacting host-guest pairs, independent of the spacer length, and independent of the unloading rate. These results indicate that the host-guest complex rupture forces were probed under conditions of thermodynamic equilibrium.