In this new method for estimating free thyroxine concentration in serum by use of polyacrylamide gel filtration, the prolonged incubation that is characteristic of equilibrium dialysis is obviated. Although the values so obtained for the percentage of free thyroxine are severalfold higher than those obtained by dialysis, the results of the two procedures correlate well over a wide range of thyroid states. In addition, the precision of the polyacrylamide method compares well with that for equilibrium dialysis. The method is convenient for use in clinical laboratories and values for free thyroxine in serum of patients with metabolic thyroid disorders are diagnostically discriminatory.