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MALDI mass spectrometric imaging is a widely-used technique for determining the spatial distribution of various compounds within biological tissues. Regardless of the application, it is advantageous to have the ability to perform profiling scans followed by subsequent MS/MS experiments to confirm the structural compositions of the analytes of interest on one platform. This work describes the imaging analysis of reserpine-dosed rat tissues using a combined MS and MS/MS workflow. Wild-type Sprague Dawley rats were orally dosed with reserpine. After two hours, the rats were sacrificed and whole body slices of 12μm thickness were mounted onto stainless-steel MALDI plates and spray-coated with MALDI matrix. MS and MS/MS imaging experiments were performed on a MALDI TOF/TOF™ instrument. MS imaging survey scans revealed that the ion signal at the calculated mass of reserpine (m/z 609.28) was distributed in the stomach and colon. Interestingly, the MS spectrum indicated a 2Da shift from the expected mass of reserpine (i.e. m/z 607.26). MS/MS imaging experiments were subsequently carried out on both precursor masses to confirm their identities. The MS/MS fragment ion pattern of the metabolite precursor with m/z 607.26 matched that of reserpine. However, as with the parent ion, several fragment ion species also displayed a 2Da shift from their expected masses. The MS/MS fragment ions co-localized with the parent ion to the same regions of the rat body. The 2Da mass shift was found to be due to a dehydration reaction at the piperidine ring nitrogen, resulting in 3,4-dehydroreserpine. MS/MS analysis of the precursor at the expected mass of reserpine (m/z 609.28) revealed that this ion was not itself reserpine but in fact an isotope of 3,4-dehydroreserpine. This work demonstrates the need for MS/MS imaging experiments alongside MS survey imaging data in order to confirm the structural identities of dosed compounds.