Thyroid hormones (THs), including triiodothyronine (T3), thyroxine (T4), and their metabolites, are essential for regulating development, growth, and energy metabolism. Thyroglobulin (Tg) produced by thyroid follicular cells acts as an essential substrate for TH synthesis. The combination of THs with Tg is a widely used serological laboratory test for thyroid function assessment. Early detection and timely intervention are significant for preventing and managing thyroid disease. In recent years, liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has emerged as a powerful tool for the precise detection of small molecular analytes and steroid hormones in clinical practice as a result of its high sensitivity and specificity. While LC‐MS/MS has been increasingly used for detecting THs and Tg recently, its application in clinical practice is still in its early stages. Recent advances in the assessment of thyroid metabolism using LC‐MS/MS in clinical samples published during 2004–2023 were reviewed, with a special focus on the use of this technique for quantifying molecules involved in thyroid diseases.