A rapid resolution liquid chromatography (RRLC) method was developed for the simultaneous determination of 23 amino acids in rat serum after pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The amino acid derivatives were separated on an Agilent Zorbax Eclipse Plus C 18 (4.6mm×50mm, 1.8μm) column at 45°C. Ultraviolet (UV) detection was set at 360nm. Good separation of 23 amino acids was achieved within 10min with a ternary gradient elution of mobile phase at a flow rate of 1.5mLmin −1 . Calibration curves were linear over the range from 1 to 500μmolL −1 with coefficients 0.9962 or better for each amino acid. The lower limits of quantification (LLOQ) of all 23 amino acids were 1μmolL −1 with signal-to-noise (S/N) ratio ≥4. Intra- and Inter-day precisions, expressed as relative standard deviation (RSD) percentages, were ranged from 0.32% to 3.09% and 0.67% to 5.82%, respectively. Finally, it was successfully applied to the determination of amino acids in rat serum with recoveries ranged from 90.8% to 106.0% and RSD percentages ranged from 1.78% to 4.68%, respectively. The results showed that the proposed method provided a shorter elution time, better resolution and sharper peak shapes for all amino acids. Compared with the conventional high performance liquid chromatography (HPLC) methods, even some ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), the established RRLC method was superior performance.