Metabolic homeostasis is critical for cell pluripotency and differentiation in human embryonic stem cells (hESCs). It has been reported that metabolic changes specifically regulate cellular signaling during hESC differentiation. This protocol describes procedures for both cell culture and detection of intracellular and extracellular metabolites in hESCs by liquid chromatography-mass spectrometry. Metabolites in glycolysis, citric acid cycle, pentose phosphate pathway, and other metabolic processes can be detected using this approach. For complete details on the use and execution of this protocol, please refer to Song et al., (2019), Yang et al., (2019), Meng et al., (2018), and Chen et al., (2011b).