We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), diiodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water–acetonitrile–acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell–Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV). This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0·5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine. Journal of Endocrinology (1997) 153, 99–104