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Although immunoassays are the more widely used method, mass spectrometry (MS)-based methods have proven to be more sensitive, specific, and reliable. Advances in MS technology and its applications for therapeutic hormone monitoring have been significant, hence integration of these methods in the clinical setting is desired. Here, we provide a general overview of HT and ART, and the immunoassay and MS-based methods currently utilized for monitoring sex hormones. Additionally, we highlight recent advances in MS-based methods and discuss future applications an