Abstract: In this work, the analytical method for the determination of amino acids by the reversed-phase separation of 2,4-dinitrofluorobenzene-amino acid derivatives has been improved in two aspects: one is the use of triethylamine phosphate(pH 2.75 and 6.50)/ACN mobile phase with high buffer capacity instead of the acetate/ACN system, another is the operation details of derivatization is stressed. The new developed method is superior to the previous one in linearity, accuracy and precision. With an injection containing Arg, Ser, Asp, Glu, Thr, Gly, Ala, Pro, His, Met, Val, Trp, Phe, Leu, Ile, Lys and Tyr as the target sample, the method has been validated to show that its linearity, accuracy and precision are ≧0.9999(≧0.9998 for Glu, Lys and Tyr), 100±1% and ≦0.5%, respectively. Without the requirement of special AA analyzer, the method is applicable to the analysis of amino acid injections and bulk drugs in the general LC laboratory.