Modern proteomic methodologies have significantly improved the possibilities of large-scale identification of proteins. However, these methodologies are limited by their inability to reliably detect endogenously expressed peptides. We describe a novel approach of combining sample preparation, comprising focused microwave irradiation and mass spectrometric peptide profiling that has enabled us to simultaneously detect more than 550 endogenous neuropeptides in 1 mg of hypothalamic extracts. Automatic switching tandem mass spectrometry and amino acid sequence determination of the peptides showed that they consist of both novel and previously described neuropeptides. The methodology includes virtual visualization of the peptides as two- and three-dimensional image maps. In addition, several novel and known post-translational modifications of the neuropeptides were identified. The peptidomic approach proved to be a powerful method for investigating endogenous peptides and their post-translational modifications in complex tissues such as the brain. It is anticipated that this approach will complement proteomic methods in the future.