Abstract: The present invention provides an improved assay for measuring thyroxine uptake by thyroxine binding globulin in which thyroxine binding globulin (TBG) activity is measured directly. The method comprises combining a sample in an aqueous solution with an enzyme donor (ED) conjugated to an analogue of polyiodothyronine that competes with thyroxine for thyroxine binding globulin binding sites. An enzyme acceptor (EA) characterized by providing a modulated enzyme activity in relation to the amount of TBG activity is combined with an enzyme donor and sample in an aqueous solution. The amount of enzyme activity in comparison to a control solution having a known amount of available TBG binding sites is determined.