Background: While T4, T3 and rT3 have routine methods available for patients with (suspicion of) thyroid disease, appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. Effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed including associated reference intervals. Methods: CLSI guidelines, EMA guidelines and the NIST protocol were used for the method validation and reference intervals. Reference intervals were determined in 253 healthy individuals (132 male, 121 female). Serum samples were deproteinized with acetonitrile followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight ¹³C₆-internal standards Results: The analytical method validation was performed for all nine THMs. Reference intervals (2.5^th to 97.5^th percentile) were determined for T0 (4.9 to 11.3 ng/dL), 3-T1 (0.06 to 0.41 ng/dL), 3,5-T2 (<0.13 ng/dL), 3,3’-T2 (0.25 to 0.77 ng/dL), T3 (66.4 to 129.9 ng/dL), rT3 (15.0 to 64.1 ng/dL), T4 (4.3 to 10.0 µg/dL), TA3 (not detected) and TA4 (2.2 to 27.2 ng/dL). Conclusions: We developed a novel, sensitive and selective LC-MS/MS method to measure seven iodothyronines and two iodothyroacetic acids in a single analysis in human serum.  A broad dynamic concentration range is observed between the nine THMs from 0.06 ng/dL to 10.0 μg/dL. This method can be of great value to better understand the clinical relevance of other THMs as well as to understand thyroid hormone metabolism in health and disease.