A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the human tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-osteoprotegerin (OPG) complex was developed by utilizing a monoclonal antibody that recognizes human soluble TRAIL as an immobilized capture component and anti-human OPG immunoglobulin G (IgG) labeled with biotin. We could quantify the TRAIL-OPG complex level (detection limit: 10 pmol/L). Employing this assay system, we demonstrated that the TRAIL-OPG complex was constitutively present in the human osteosarcoma cell lines, HOS, MG-63, and human salivary gland adenocarcinoma cell line, HSG.