Therapeutic peptides are a fast-growing class of pharmaceuticals. Like small molecules, the costs associated with their discovery and development are significant. In addition, since the preclinical data guides first-in-human studies, there is a need for analytical techniques that accelerate and improve our understanding of the absorption, distribution, metabolism, and excretion (ADME) characteristics of early drug candidates. Mass spectrometry imaging (MSI), which can be used to visualize drug distribution in intact tissue, has been extensively used to study small molecule drugs, but only applied to a limited extent to larger molecules, such as peptides, after dosing. Herein, we use MSI to obtain spatial information on the distribution and metabolism of a peptide drug. The immunosuppressant cyclosporine (CsA), a cyclic undecapeptide, was used as a-proofof-concept peptide and investigated by desorption electrospray ionization (DESI) MSI. Calibration curves were made based on a spiked tissue homogenate model. Different washing protocols were tested to improve sensitivity, but CsA, being a quite lipophilic peptide, was found not to benefit from tissue washing. The distribution of CsA and its metabolites were mapped in whole-body mouse sections and within rat organs. Whole-body DESI-MSI studies in mice showed widespread distribution of CsA with highest abundance in organs like the pancreas and liver. After 24 h, hydroxy and dihydroxy metabolites of CsA were detected predominantly in the intestines, which were largely devoid of CsA. In addition to the DESI-MSI experiments, MALDI-MSI was also conducted on rat jejunum at higher spatial resolution, revealing the morphology of the jejenum at greater detail; however, DESI provided similar results for drug and metabolite distribution in rat jejunum at apparent slightly better sensitivity. Given its label-free nature, MSI could provide valuable ADME insight, especially for candidates in the early-stage pipeline before radiolabeling.