Objective: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) was used to establish quantitative methods for 16 amino acids. Accurate quantitative analysis of plasma and urine samples was achieved. Methods: Using UPLC-MS technology, the liquid phase was gradient elution with 20 mmol·L-1 ammonium formate aqueous solution (A) and acetonitrile (containing 15%20 mmol·L-1 ammonium formate aqueous solution) (B) as the mobile phase, and the mass spectrometry was detected in multiple reaction monitoring mode (MRM). The specificity, limit of detection, limit of quantification, linearity, accuracy, precision, residual effect and stability were evaluated. The method was applied to the quantification of amino acids in plasma and urine of mice. Results: The results showed that the method met the relevant standards of methodology verification. The results of accurate quantification of amino acids in plasma and urine of mice showed that this method could simultaneously detect and quantify 16 amino acids in plasma and urine of mice. Conclusion: A non-derivatized method for accurate quantification of 16 amino acids in mouse plasma and urine has been established by UPLC-MS technology with good precision and accuracy, which provides a reference for accurate quantification of amino acids in mouse plasma and urine.