This chapter aims to provide a practical guide for the development and validation of mass spectrometry (MS) based methods for the clinical laboratory. A complete understanding of the processes involved in development, validation, and implementation of MS-based tests is crucial for broader adoption of MS platforms in clinical laboratories. While each analyte has its own unique challenges, this chapter provides a step-by-step guide covering various aspects and considerations for the development and validation of clinical LC-MS/MS methods. Development of MS-based methods should start with defining the intended clinical use, required performance characteristics, and regulatory requirements, in addition to an assessment of project feasibility. The method development process typically includes optimization of MS instrument parameters, chromatographic separation, and sample preparation, with a focus on achieving the targeted performance characteristics while ensuring specificity. In addition to the method development process, this chapter discusses preparation for and execution of the validation process. By following the outlined procedures, laboratories can develop robust, high-quality LC-MS/MS assays, which are suitable for clinical implementation and meet regulatory requirements.