The trace characterization of physiologically active substances with low molecular weight (e.g., steroids, catecholamines, prostaglandins, and oligopeptides), which are classiˆed as``haptens'', is an important subject in clinical analysis, and competitive immunoassays have conventionally been used for this purpose. However, the subfemtomole-range determination of haptens is very di‹cult, as the sensitivity of competitive immunoassays is essentially limited by the a‹ni-ty of the anti-hapten antibodies that barely reaches the range of 10 11 (l/mol) as the a‹nity constant (K a ). Although a noncompetitive``immunometric assay'' format, the two-site immunometric assay (sandwich immunoassay), enables even subattomole-range measurements of macromolecules such as proteins, this principle can not be directly applied to haptens, as their low molecular mass prohibits simultaneous binding by two antibody molecules. To overcome such limitations, we are required either to create artiˆcial antibodies showing ultrahigh a‹nity to haptens by protein engineering of antibody molecules (``antibody engineering'') or establishment of novel immunometric assay formats applicable to haptens. This review surveys the background and recent approach for subfemtomole-range determination of haptens using novel immunometric assay methods. Our studies for the development of hapten immunometric assays are also described.