Metabolic stability is defined as the percentage of parent compound lost over time in the presence of a metabolically active test system. For metabolic stability assays, the typical test systems are liver microsomes, liver S9, or hepatocytes (plated or suspended), depending on the goal of the assay. To determine the metabolic stability of a new chemical entity, quantification of the drug candidate from incubate supernatants is required and usually accomplished by high-performance liquid chromatography (HPLC) with mass spectrometry. This analytical approach incorporates specificity, increased sensitivity, and higher throughput via decreased method development and analysis runtime. By understanding the metabolic stability of compounds early in discovery, compounds can be ranked for further studies, and the potential for a drug candidate to fail in development as a result of pharmacokinetic reasons may be reduced. This chapter details the procedures for performing a standard metabolic stability assay and the subsequent analysis of generated incubate samples.