Atomic force microscopy (AFM) with chemical specificity using chemically modified AFM probes, so-called chemical force microscopy, was applied to study surface chemical reactions on the nanometer scale. To overcome the typical limitations of conventional AFM in following reactions in real-time, i.e. slow data acquisition, as well as thermal and instrumental drifts, we have introduced a new approach, named inverted chemical force microscopy (iCFM). In iCFM the reactants are immobilized on the AFM tip rather than on the substrate. The chemical reactions take place at the surface of the tip and are probed in real-time by force–displacement measurements on an inert octadecanethiol-covered Au substrate. The reactions studied were the hydrolysis and aminolysis of 11,11′-dithiobis(N-hydroxysuccinimidylundecanoate) (NHS-C10). By iCFM intermolecular interactions and hence reaction kinetics can be quantitatively studied on the level of ∼10–100 molecules. In particular, our iCFM data showed that the aminolysis reaction with n-butylamine on SAMs of NHS-C10 is a spatially heterogeneous reaction. In addition, information about the defect density of reactive SAMs was obtained.