Amino acids were determined by precolumn derivatization with diethyl ethoxymethylenemalonate and reversed-phase high-performance liquid chromatography (HPLC) with spectrophotometric detection at 280 nm. The reaction time was 50 min and the derivatives were stable at room temperature. Chromatographic resolution of a mixture of the derivatives of seventeen amino acids, including proline and cystine, was achieved within 35 min using a binary gradient system. The detection limit was 3 pmol. Amino acid analyses of acid hydrolysates of two proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multi-purpose HPLC system.