Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.