Due to the high variance in available protocols on iodide-131 (<sup>131</sup>I) ablation in rodents, we set out to establish an effective method to generate a thyroid-ablated mouse model that allows the application of the sodium iodide symporter (NIS) as a reporter gene without interference with thyroidal NIS. We tested a range of <sup>131</sup>I doses with and without prestimulation of thyroidal radioiodide uptake by a low-iodine diet and thyroid-stimulating hormone (TSH) application. Efficacy of induction of hypothyroidism was tested by measurement of serum T<sub>4</sub> concentrations, pituitary TSHβ and liver deiodinase type 1 (DIO1) mRNA expression, body weight analysis, and <sup>99m</sup>Tc-pertechnetate scintigraphy. While 200 µCi (7.4 MBq) <sup>131</sup>I alone was not sufficient to abolish thyroidal T<sub>4</sub> production, 500 µCi (18.5 MBq) <sup>131</sup>I combined with 1 week of a low-iodine diet decreased serum concentrations below the detection limit. However, the high <sup>131</sup>I dose resulted in severe side effects. A combination of 1 week of a low-iodine diet followed by injection of bovine TSH before the application of 150 µCi (5.5 MBq) <sup>131</sup>I decreased serum T<sub>4</sub> concentrations below the detection limit and significantly increased pituitary TSHβ concentrations. The systemic effects of induced hypothyroidism were shown by growth arrest and a decrease in liver DIO1 expression below the detection limit. <sup>99m</sup>Tc-pertechnetate scintigraphy revealed absence of thyroidal <sup>99m</sup>Tc-pertechnetate uptake in ablated mice. In summary, we report a revised protocol for radioiodide ablation of the thyroid gland in the mouse to generate an in vivo model that allows the study of thyroid hormone action using NIS as a reporter gene.