In this study, a robust, selective and simplified method was developed and validated for the simultaneous quantitative analysis of 23 underivatized amino acids in human serum using mixed-mode chromatography with tandem mass spectrometry (LC–MS/MS). Serum samples were deproteinized with acetonitrile and subjected to LC–MS/MS analysis. The chromatographic separation of amino acids was achieved using a mixed-mode column (150 × 3 mm, 3 μm) with a gradient elution system; the mobile phase consisted of 50 mM ammonium formate and 0.1% formic acid in acetonitrile. The total run time was 22 min. Eluted compounds were detected in the electrospray ionization-positive mode with multiple reaction monitoring. The validation study evaluated linearity, repeatability, intra and inter-day accuracy and precision, and matrix effect. The validation results were satisfactory in all the tested parameters. This method was successfully applied to the analysis of amino acids in the clinical sample of human serum.