Biogenic amines are critically important neuromodulators in both vertebrates and invertebrates. Quantification of these amines can be difficult, particularly in neural extracts of Drosophila melanogaster that contain interfering electroactive compounds. We have developed a method for the reliable separation and quantification of the biogenic amines dopamine, serotonin, tyramine, and octopamine in Drosophila brain extracts using high performance liquid chromatography with electrochemical detection. Our method obviates the need for complex preparatory procedures or instrumentation, and can reproducibly detect picogram quantities of these amines. By optimizing the composition of the mobile phase and the electrode potential, and by examining common complications in the analysis of biological samples, we have developed a reliable technique for monitoring levels of biogenic amines in the Drosophila brain.