A new method for the separation and quantification of primary and secondary amino acids by reversed-phase high-performance liquid chromatography and spectrophotometric detection is described. The use of the novel derivatizing reagent N,N-diethyl-2,4-dinitro-5-fluoroaniline yields derivatives that are more stable to light and heat in solution than the traditional ones. A simple gradient between acidic acetate buffer and acetonitrile allows the complete separation of 21 amino acids in 80 min at room temperature. The good reproducibility of peak areas and retention times allows the application of this simple and low-priced method to the determination of amino acids in the range 50-500 pmol per residue.