Anuran metamorphosis involves the transformation of an aquatic tadpole into a juvenile frog. This process is completely dependent upon thyroid hormones (THs). Although much research has been focused on changes in gene expression programs during this postembryonic developmental period, transitions in the metabolic profiles are relatively poorly understood. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) is a technique that generates highly multiplexed mass spectra while retaining spatial location information on a thin tissue section. Reconstructed ion heat maps are correlated with morphology of the tissue section for biological interpretation. The present study is the first to use whole-body MALDI-MSI on tadpoles to gain insights into anuran metamorphosis. Approximately 1000 features were detected in each of five tissues examined (brain, eye, liver, notochord, and tail muscle) from premetamorphic North American bullfrog (Rana [Lithobates] catesbeiana) tadpoles. Of these detected metabolites, 1700 were unique and 136 were significantly affected by exposure to 50 nM thyroxine for 48 h. Of the significantly-affected metabolites, 64 features were tentatively identified using the MassTRIX annotation tool. All tissues revealed changes in lipophilic compounds including phosphatidylcholines, phosphatidylinositols, phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylserines. These lipophilic compounds made up the largest portion of significantly-affected metabolites indicating that lipid signaling is a major target of TH action in frog tadpoles.