Supported model catalysts exclusively exposing monomeric vanadyl species were prepared by washing V2O5/TiO2 samples (Eurocat EL10VL and EL10V8) with aqueous solutions of ammonia. FT-NIR Raman spectroscopy confirmed that V2O5 crystallites and polymeric species which had been present on both samples were removed by the washing treatment, leaving behind monomeric vanadyl species. The adsorption of toluene was studied using in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) in order to elucidate the role of monomeric vanadyl species in hydrocarbon activation and transformation. Oxidative adsorption occurred in all samples, suggesting monomeric vanadyl species to be crucial for this step. Toluene oxidation had proceeded to a lower extent on the washed samples than on the unmodified Eurocat samples which is consistent with the capability of fast oxygen insertion associated with polymeric vanadia species and V2O5 crystallites. These species also proved to be essential to make gas-phase oxygen available to adsorbed hydrocarbons.