Identification of low-abundance, low-molecular-weight native peptides using non-tryptic plasma has long remained an unmet challenge, leaving potential bioactive/biomarker peptides undiscovered. We have succeeded in efficiently removing high-abundance plasma proteins to enrich and comprehensively identify low-molecular-weight native peptides using mass spectrometry. Native peptide sequences were chemically synthesized and subsequent functional analyses resulted in the discovery of three novel bioactive polypeptides derived from an epidermal differentiation marker protein, suprabasin. SBSN_HUMAN[279-295] potently suppressed food/water intake and induced locomotor activity when injected intraperitoneally, while SBSN_HUMAN[225-237] and SBSN_HUMAN[243-259] stimulated the expression of proinflammatory cytokines via activation of NF-κB signaling in vascular cells. SBSN_HUMAN[225-237] and SBSN_HUMAN[279-295] immunoreactivities were present in almost all human organs analyzed, while immunoreactive SBSN_HUMAN[243-259] was abundant in the liver and pancreas. Human macrophages expressed the three suprabasin-derived peptides. This study illustrates a new approach for discovering unknown bioactive peptides in plasma via the generation of peptide libraries using a novel peptidomic strategy.