Iodotyrosine deiodinase (DEHAL1) is a crucial enzyme in iodine homeostasis. Unbound mono- and diiodotyrosines are indispensable byproducts of thyroid hormone biosynthesis. Their iodine needs to be recovered to avoid iodine deficiency, as observed in genetic defects in DEHAL1. Despite its importance, the enzyme is rarely studied. The deiodination process can be monitored by radioactive tracers or via techniques involving mass spectrometry. However, isotope-labeled molecules are expensive, not always commercially available, and their use is legally restricted, whereas mass spectrometry requires sophisticated, costly, and sensitive instrumentation. To circumvent these difficulties, we adapted the nonradioactive iodothyronine deiodinase assay to determine DEHAL1 activity by a colorimetric readout, based on the Sandell-Kolthoff reaction. DEHAL1 was recombinantly expressed and used to optimize the assay in microtiter format. We applied the setup to scenarios of alternative substrate screening or search for compounds potentially acting as endocrine disrupting compounds, without identifying novel readily accepted substrates or inhibitors yet. Next, the assay was adapted to ex vivo material, and activity was reliably determined from rodent kidney and other tissues. Analyzing two mouse models of hyperthyroidism, we observed a decreased renal Dehal1 activity and mRNA expression. Our results show that this nonradioactive DEHAL1 assay is suited to screen for potential endocrine disrupters and to monitor endogenous Dehal1 expression. We harmonized the assay protocols to enable iodothyronine deiodinase and DEHAL1 activity measurements from the same samples. Hereby, a more complete view on iodine metabolism by these predominant deiodinating activities can be obtained from a given sample by a similar process flow.