Mass spectrometry-based assays play a pivotal role in modern diagnostics, yet their regulation remains complex and dynamic. Mass spectrometry assays are predominantly laboratory developed tests (LDTs), which have been governed under the Clinical Laboratory Improvement Amendments (CLIA) by the Centers for Medicare & Medicaid Services (CMS). The recent Food and Drug Administration (FDA) final rules have introduced expanded oversight on LDTs in general and mass spectrometry-based assays are impacted as a result. This chapter explores the regulatory requirements for these tests, highlighting CLIA standards, the evolving FDA framework, and their implications for laboratories. By addressing compliance and operational challenges, the chapter aims to demonstrate the delicate balance of encouraging innovation and ensuring quality, as well as to guide laboratories in leveraging mass spectrometry while adhering to the latest regulatory mandates.