Passivated substrates are provided for use in assays, comprising at least one covalently bonded ligand having specific binding activity for a molecule, and at least one covalently bonded blocking agent, wherein said ligand is directly bonded to the substrate surface. In certain embodiments, the ligand and blocking agent are covalently bonded only to the substrate surface, and not directly bonded to each other. In certain other embodiments, the ligand and blocking agent have at least one additional covalent bond to one another. Methods for preparing and using passivated substrates in bioassays are also provided.