Abstract: Objects: To evaluate the quality of the root of Ficus hirta Vahl, a pre-column derivation-high performance liquid chromatography (HPLC) method was established for the determination of 16 kinds of amino acids in the root of Ficus hirta from different origins and leaf shapes. Methods: The contents of 16 kinds of amino acids in Ficus hirta were determined by HPLC with phenyl isothiocyanate pre-column derivatization method. The separation was achieved using a ultimate amino acid column (4.6 mm×250 mm, 5 μm) under the gradient elution with the mobile phases composed of 0.1 mol/L sodium acetate buffer and acetonitrile at the flow rate of 1.0 mL/min, column temperature of 40 ℃ and UV detector wavelength at 254 nm. Results: The contents of 16 kinds of amino acids had a good linear relationship, and the correlation coefficients was more than 0.9992, while the average recovery rates (n=6) were 98.1%~105.9% and RSD were 0.67%~2.11%. The average contents of L-asparaginic acid, L-glutamic acid, L-serine, glycine, L-histidine, L-arginine, L-threonine, L-alanine, L-proline, L-tyrosine, L-valine, L-methionine, L-isoleucine, L-leucine, L-phenylalanine, L-lysine were respectively 3.308, 3.424, 2.750, 2.978, 1.302, 4.549, 2.778, 3.989, 3.818, 1.327, 3.732, 0.395, 2.925, 4.549, 2.873, 3.305 mg/g. There were differences in the content of total amino acid in Ficus hirta from different leaf shapes. Compared with leaf shapes, the three-finger shapet had the highest total amino acid, followed by seven-finger shpape, and then five-finger shape, and the last was undivided. The content of total amino acid in Ficus hirta from different producing areas were also different. The highest content was in Lingshan county of Qinzhou city of Guangxi province, followed by Yuannan town of Heyuan city of Guangdong province, and the lowest content was in Chengxi town of Longhai district of Zhangzhou city of Fujian province. Conclusion: 16 kinds of amino acids were separated by this method, which was reliable, accurate and had high recovery rate. It could be used for the determination of amino acids in Ficus hirta, and the content of amino acids could be used to evaluate the quality of Ficus hirta.