Neuropeptides regulate most, if not all, biological processes in the animal kingdom, but only seven have been isolated and sequenced from Drosophila melanogaster. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomics approach aims at the simultaneous identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their posttranslational modifications. Using nanoscale liquid chromatography combined with tandem mass spectrometry and data base mining, we analyzed the peptidome of the larval Drosophila central nervous system at the amino acid sequence level. We were able to provide biochemical evidence for the presence of 28 neuropeptides using an extract of only 50 larval Drosophila central nervous systems. Eighteen of these peptides are encoded in previously cloned or annotated precursor genes, although not all of them were predicted correctly. Eleven of these peptides were never purified before. Eight other peptides are entirely novel and are encoded in five different, not yet annotated genes. This neuropeptide expression profiling study also opens perspectives for other eukaryotic model systems, for which genome projects are completed or in progress.