An improved chromatographic procedure for the quantitative determination of iodoamino acids in thyroglobulin is described. In this method, thyroglobulin hydrolyzed by the combined use of Pronase and aminopeptidase M is applied to a column of AG 50W-X4 (30–35μ) equilibrated with 0.04 m ammonium acetate buffer, pH 4.7, containing 30% (v/v) ethanol. By the application of a simple gradient of increasing pH to IN NH4OH in the presence of 30% ethanol, five components, i.e., I−, mono-iodotyrosine, diiodotyrosine, thyroxine, and 3,5,3′-triiodothyronine are separately eluted in that order, and they are determined quantitatively by automatic analysis of the effluent based on the ceric-arsenite reaction. Reproducible and accurate data are thus provided within 3 hr; the amount of undigested material is almost nil and the extent of deiodination of iodoamino acids occurring during the operation can be minimized.