A new HPLC method has been established for determination of 3-monoiodotyrosine (MIT), 3,5-diiodotyrosine (DIT), 3,5-diiodothyronine (T2), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3), and thyroxine (T4) produced by hydrolysis of iodinated casein with barium hydroxide. The hydrolytic stability of each analyte was evaluated. Iodinated casein was hydrolyzed with saturated barium hydroxide solution for 16 h at 110°C and the barium ions were then removed as barium sulfate. Reversed-phase HPLC was performed on a 2.1 mm × 150 mm, 5 μm particle, C18 column with a mixture of acetonitrile and 0.1% (v/v) formic acid as mobile phase at a flow rate of 0.2 mL min–1. Acetonitrile was maintained at 5% (v/v) for 5 min and then increased linearly to 50% (v/v) within 35 min. All analytes were quantified by measuring the absorbance at 280 nm. Validation data indicated the method was linear, with regression coefficients (R2) > 0.998, in the concentration ranges investigated. Sensitivity was adequate—limits of detection (LOD) were 0.04–0.38 μg mL–1 and limits of quantification (LOQ) were 0.05–0.38 μg mL–1. Accuracy and precision were acceptable — for all the analytes recovery was 82.0–93.0% and repeatability, as relative standard deviation, was 1.0–3.0%. Hydrolytic stability tests indicated MIT and DIT are much more stable than the other analytes. rT3 was not released directly from iodinated casein but was formed by deiodination of T4 during hydrolysis. The method could be used to identify iodinated casein, to evaluate its activity and quality, and for supervision and regulation of feed additives.