Updated project metadata. To identify and quantify the relative abundances of cell-cell signaling peptides in different physiological states, LC-MS-based peptidomics workflows are commonly utilized on freshly dissected tissue. In such animal experiments, the administration of general anesthetics is an important step for many research projects. However, acute anesthetic administration may rapidly change the measured abundance of transmitter molecules and metabolites which may confound experimental results. The aim of this study was to evaluate the effect of short-term (< 5 min) anesthetic administration on the measured abundance pituitary peptides, as evaluated by a typical peptidomics workflow. To accomplish this goal, we compared peptide abundances in the rat pituitary following administration of 5% isoflurane, 200 mg/kg sodium pentobarbital, or no anesthetic administration using a label-free peptidomics protocol.