Abstract: A method of determining the concentration of the free portion of a ligand present in a biological fluid which also contains the ligand bound to one or more natural binders involves the single step of mixing a sample of the fluid with a labelled derivative of the ligand and a specific binder for the ligand, incubating the mixture, and determining the proportion of the labelled derivative of the ligand bound to the specific binder. The labelled derivative of the ligand is chosen to bind strongly to the added specific binder, but to bind not at all, or much more weakly than does the ligand, to the natural binders in the biological fluid. A small amount of the labelled derivative can be used, and the binding equilibrium of the ligand is not appreciably perturbed. Ligands include thyroxine, tri-iodothyroxine and cortisol.