Abstract: Research and development of 3,3',5-Triiodo-L-thyronine (T3) purity reference material is a way to establish the primary material for clinical measurement value traceability, which can provide more accurate and comparable evidence in thyroid disease diagnosis. In the mass balance quantitative approach, determining and analyzing those structural-related impurities is essential. Such investigation could also be an effective way to reduce the uncertainty of the standard value. First, nuclear magnetic resonance and high-resolution mass spectrometry analytical tools have been employed to validate the chemical structure of the reference material. Then, the structural-related impurities are separated and qualitatively analyzed by using reliable analytical technologies, including high-performance liquid chromatography, high-resolution mass spectrometry, and ion mobility spectrometry. The results indicated that there are three kinds of structural-related impurities involved in the T3 purity reference material (NIM-RM3239), which are Diiodo-L-thyronine, Chloro-diiodo-L-thyronine, and L-Thyroxine. The relative content of each impurity is greater than 0.1% in UV 220nm. Furthermore, the chiral isomer separation result indicated that no dextroisomer (D-T3) existed in NIM-RM3239. These analytical results can be utilized as the foundations in the mass balance approach, applied in developing T3 primary certified reference material.