Background: In contrast to the thyroid hormones (TH) 3,3',5-triiodothyronine (T3) and thyroxine (T4), current literature on thyroid hormone metabolite concentrations in the hypothyroid and hyperthyroid states is inconclusive. It is unknown how thyroidectomy affects thyroid hormone metabolite concentrations and if levothyroxine (LT4) replacement therapy after thyroidectomy restores thyroid hormone metabolite concentrations in those without a thyroid gland. The treatment of patients with differentiated thyroid cancer (DTC) covers the euthyroid, hypothyroid, and (subclinical) hyperthyroid states and therefore provides a unique model to answer this. Here, we prospectively studied nine TH and its metabolites (THM) across different thyroid states in a cohort of patients treated for DTC. Also, three potentially important determinants for THM concentrations were studied. Methods: We prospectively included patients aged 18 to 80 years who were scheduled for DTC treatment at the Erasmus MC. Peripheral blood samples were obtained before surgery (euthyroid, endogenous TH production), after surgery just before radioactive iodine therapy (hypothyroid), and six months later on LT4 therapy ([subclinically] hyperthyroid, exogenous T4 supplementation). Nine THMs were quantified in serum with an established liquid chromatography/tandem mass spectrometry method. Repeated measurement analysis was used to compare the three different thyroid states with each other for each THM, while linear regression was used to determine the association between THM concentrations and age, sex, and kidney function. Results: In total, 77 patients (mean age 49 years; 65% women) were eligible for the study. 3,5-diiodothyronine and 3,3',5-triiodothyroacetic acids were below the lower limit of detection. Compared with the euthyroid state, all THMs were significantly decreased in the hypothyroid state and significantly increased in the (subclinically) hyperthyroid state, with T3 concentrations remaining within the reference interval. Higher age was associated with higher 3-monoiodothyronine (3-T1) concentrations (p < 0.001). Women had higher L-thyronine concentrations than men (p = 0.003). A better kidney function was associated with lower 3-T1 concentrations (p < 0.001). Conclusions: All THMs decrease after a thyroidectomy and increase under thyrotropin (TSH)-suppressive LT4-therapy, suggesting that formation of thyroid hormone metabolites is dependent on peripheral extrathyroidal metabolism of T4. This is also reflected by T3 concentrations that remained within the reference interval in patients receiving TSH-suppressive LT4-therapy as T3 has some thyroidal origin.