Metabolites are small molecules crucial for metabolic processes, while their analogs are structurally similar compounds used to study and manipulate these pathways in medicine and science. Metabolite analogs serve as powerful tools in drug development, research, and therapeutics by mimicking or inhibiting natural metabolites to alter biochemical pathways, impacting both catabolism and anabolism. These analogs are pivotal in research and treatment, influencing pathways like catabolism and anabolism. Common metabolites like glucose, amino acids, and nucleotides play vital roles in pathways such as PTEN and mTOR, which are essential for regulating cell growth, proliferation, and survival. By influencing these pathways, metabolite analogs can help elucidate metabolic mechanisms and offer potential treatments for diseases like cancer and metabolic disorders. This protocol chapter will detail the preparation and handling of metabolites, specifically focusing on a PTEN inhibitor. We will outline the steps for dissolving and storing these compounds, followed by a comprehensive protein extraction protocol. Finally, the use of Proteome Discoverer software will be discussed to analyze the resultant proteome changes.