Abstract: To establish a methodology for detecting the amino acid content in rice peptides, the research employed the pre-column derivative phenyl isothiocyanate (PITC) for realizing simultaneous, rapid, and effective analysis of 16 amino acids, including aspartate (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), and lysine (Lys). The analysis was carried out using an Athena AAA chromatographic column as the analysis column, with a flow rate of 1.0 mL/min, column temperature of 35 ℃, and UV detector wavelength of 254 nm. A gradient elution was performed with a mobile phase consisting of methanol:acetonitrile:water=20:60:20 and 50 mmol/L sodium acetate (pH6.5). The results revealed a strong linear relationship among the 16 amino acids within the range of 0.125 to 2.5 μmol/mL, with a coefficient of determination R² ≥0.9999 for 14 amino acids. The remaining 2 amino acids exhibited coefficient of determination R² were 0.9998 and 0.9994, respectively. The detection limit ranged from 0.003% to 0.018%, while the quantitative limit ranged from 0.010% to 0.059%. The spiked recovery rate ranged from 82.21% to103.59%, and the relative standard deviation (RSD) was less than 5% for all measurements. This method effectively separates the 16 amino acids in rice peptides, with glutamic acid (Glu) having the highest content ranging from 13.11%±0.39% to 21.38%±0.48%. The amino acids with the lowest content are histidine (His) or methionine (Met), ranging from 1.59%±0.05% to 2.27%±0.03% or 1.26%±0.05% to 2.32%±0.06%, respectively. The experimental method employed in this study demonstrates high accuracy, precision, sensitivity and recovery rate, thereby, indicating its suitability for analyzing the amino acid content in rice peptides.