The concentrations of hydroxyl groups located inside and on the surface oxide films of a commercially pure titanium, cp-Ti, a type 316L austenitic stainless steel, SS, and a cobalt-chromium-molybdenum alloy, Co-Cr-Mo, were evaluated using X-ray photoelectron spectroscopy, XPS, and a zinc-complex substitution technique. As a result, the concentrations of the hydroxyl groups detected by the zinc-complex substitution technique, defined as active hydroxyl groups, were much larger than those detected by other conventional techniques. The concentration of the active hydroxyl groups on Co-Cr-Mo was significantly larger than those on cp-Ti and SS. Poly(ethylene glycol), PEG, is a biofunctional molecule that inhibits the adsorption of proteins. The immobilization of PEG to metal surfaces by electrodeposition or immersion is an important technique to biofunctionalize the metals. The amounts of the PEG layer immobilized on the metals were governed by the concentrations of the active hydroxyl groups on each surface oxide in the case of electrodeposition; it was governed by the relative permittivity of the surface oxide in the case of immersion. The estimation of active hydroxyl groups on the surface oxide film with the zinc-complex substitution technique is useful for the elucidation of reactions between metal substrates and immobilized molecules.