This paper describes a reliable and rapid method for the complete separation and quantitation of twenty-five amino acids typically found in plants, based on reversed phase high-performance liquid chromatography-linked fluorescence detector using a 150 x 4.6 mm Zorbax Eclipse AAA column. Plant tissue free amino acids (FAA) were extracted by ultrasonication with 5% (v/v) aqueous trifluoroacetic acid followed by ultrafiltration of extracts. The following analysis of amino acids was performed through programmed precolumn derivatization with ortho-phthalaldehyde and 9-fluorenylmethyl chloroformate reagents and efficient elution of derivatives within 26 min using binary gradient scheme. The method was validated over a concentration range of 4.5-450 mol L~(-1) (M). Separation analysis showed good selectivity (resolution > 1.5) for most amino acids. The average repeatability(RSD%,relative standard deviation) of the analysis at seven calibration concentrations was below 4% and ranged from 1.13% to 12.04%. The intra-day mean coefficient of variation at two concentrations (22.5 and 90 M) was within 2%, and the intermediate precision was less than 4%. The limits of detection were between 0.012 and 6.68 M. The coefficients of determination (R~2) of the linear calibration curves were from 0.9989 to 0.9999. When the method was applied to plant samples,the FAA recoveries at two spiked levels (25 and 100 M) ranged from 67.0% to 108.9% with an average of 94.4%, and the precision was 0.26%-12.31% RSD. A specific application combining this method with optimized extraction and interference removal procedures was successfully used to determine the FAA pools in different plant tissues. Finally, a PLS-DA multivariate statistics model was validated for the classification of three plant species according to their FAA profiles.