A diazoketo-functionalized alkoxysilane compound was synthesized, and its self-assembled monolayer (SAM) formation was studied on glass and silicon substrates. Infrared spectroscopy was employed to follow the photoreaction in this system, which proved that carboxylic groups were generated front diazoketo groups in the surface of the substrate. Photopatterning of biotin/streptavidin on the diazoketo-functionalized SAM shows the potential of this novel platform for biomolecular patterning.