Aim: Branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) were suggested as potential biomarkers in liver disease. This study aimed to develop and validate a simple and rapid LC-MS/MS method to simultaneously measure serum BCAAs and AAAs levels in patients with liver injury, and further establish reference intervals of Chinese healthy adult populations.Patients & methods: Samples were prepared by a one-step protein precipitation and analysis time was 4min per run.Results: The validation results showed good linearity (r2>0.9969), satisfactory accuracy (94.44%-107.75%) and precision (0.10%-5.90%).Conclusion: This method proved to be suitable for high-throughput routine clinical use and could be a valuable adjunct diagnosis tool for liver injury and other clinical applications.