Metabolomics is becoming increasingly important as it provides a comprehensive analytical platform to better understand the biological functioning of a cell or organism. In recent years, microbial metabolomics has received much attention in research areas from new drug discovery to metabolic engineering. An efficient and accurate method to measure the intracellular metabolites of a specific microbial species is a key prerequisite for metabolome analysis. In this study, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. A filter-based bacteria sampling system was utilized to separate the media and bacteria; fast quenching with nitrogen was applied to prevent any metabolite leakage; a glass beads beater was used for intracellular metabolite extraction; and the LC-QTOF was combined to quantify the intracellular amino acids of S. aureus. This protocol is demonstrated to be an efficient method for analyzing the intracellular metabolites of S. aureus.