The cumulative exposure of chronic, low doses of ionizing radiation is a concern of for human health. Prior studies have focused on the analysis of individual organs or biofluids (sp. blood) to determine biomarkers of exposure. This study is the first to apply mass spectrometry imaging to experimental animals that have been exposed to different doses of ionizing radiation over an extended period of time. To determine an optimal analytical method, experimental conditions including the selection of MALDI matrix and mass analyzer were examined and later applied to an experimental cohort of Medaka fish, a vertebrate model for both human and ecological aspects of radiation exposure.