The catalytic upgrading of biomass is an important step toward realizing renewable chemical production. Pt/WOx and the inverse catalyst, WOx/Pt, have been shown to be highly active and selective for the C–O bond scission of various biomass-derived oxygenates. Yet, the nature of the active sites and detailed reaction mechanisms have not been well understood. In this study, carbon monoxide and isopropyl alcohol (IPA) have been used as probe molecules to study the active sites of WOx/Pt(111) model surfaces. Temperature-programmed desorption experiments identified two distinct active sites responsible for different reaction pathways: dehydrogenation over Pt sites and dehydration over WOx sites. High-resolution electron energy loss spectroscopy (HREELS) identified the surface reaction intermediates. In situ infrared reflection absorption spectroscopy and HREELS measurements confirmed the presence of hydroxyl groups on WOx, which were consumed upon the adsorption of IPA, suggesting that the hydroxyl groups were the active sites for IPA dehydration. Density functional theory (DFT) calculations further revealed the reaction mechanisms and activation barriers of the IPA dehydrogenation and dehydration reactions. The comparison of different in situ-generated hydroxyl groups on WOx by DFT calculations suggested that the most active sites for dehydration should be those with protons least strongly bound (site formation energy of −0.3 eV), with a dehydration activation barrier of 1.23 eV. The understanding from this study provides insights into the design of relevant metal/metal oxide catalysts for the upgrading of biomass-derived oxygenates.