Amino acids in cells are used not only as building blocks for protein synthesis but also for other purposes such as signal transduction and ATP synthesis. For example, amino acids are essential signaling molecules to activate mTORC1 (mechanistic target of rapamycin complex 1), a serine/threonine kinase complex, which plays a key role in the regulation various biological processes including lipid synthesis, translation, autophagy and so on. Also, dysregulation of amino acid homeostasis is often implicated in the pathogenesis of cancer, diabetes and neurodegenerative disorder. Amino acids in cell culture medium are supposed to significantly affect the intracellular amino acid contents, and subsequently, amino acid-related cellular functions. However, the relationship between amino acid availability and intracellular amino acid concentration is still poorly understood. In this study, we established a reversed-phase HPLC method to quantitatively analyze the amino acids in cell lysates. Among 20 proteinogenic amino acids, 19 amino acids except tryptophan was successfully detected. By manipulating the amino acid availability in mammalian cell culture, the effects on the intracellular free amino acid concentration were further studied.