Characterizing the orientation of covalently conjugated proteins on nanoparticles, produced for in vitro and in vivo targeting, though an important feature of such a system, has proved challenging. Although extensive physicochemical characterization of targeting nanoparticles can be addressed in detail, relevant biological characterization of the nanointerface is crucial in order to select suitable nanomaterials for further in vitro or in vivo experiments. In this work, we adopt a methodology using antibody fragments (Fab) conjugated to gold nanoparticles (immunogold) to map the available epitopes on a transferrin grafted silica particle (SiO2-PEG8-Tf) as a proxy methodology to predict nanoparticle biological function, and therefore cellular receptor engagement. Data from the adopted method suggest that, on average, only ∼3.5% of proteins grafted on the SiO2-PEG8-Tf nanoparticle surface have a favorable orientation for recognition by the cellular receptor.