Abstract: There is described an assay system for the assessment of unoccupied specific ligand binding sites on proteins in biological fluids. The assay is carried out with a multilayer assay element and is based on the principle of equilibrium distribution in which a ligand distributes itself between unoccupied sites on the proteins and the remainder of the element. The ligand can be labeled with a detectable moiety or it may be unlabeled and detected through the use of another detectable species. In one embodiment the assay format is utilized to estimate the number of sites on thyroxine binding globulin (TBG) which are not occupied by thyroxine (T4) or triiodothyronine (T3).