Imaging mass spectrometry (IMS) requires the application of special sample pretreatment methods to enable the visualization of the target compounds with high sensitivity and high resolution. The ionization efficiency of matrix-assisted laser desorption/ionization is particularly low when used with IMS to detect steroid hormones. Additionally, steroid hormones cannot be analyzed with existing IMS pretreatment methods. This report describes an on-tissue derivatization method that enables the visualization of corticosterone and its successful IMS analysis with high sensitivity and high resolution using an iMScope TRIO microscope. Moreover, we describe the identification of the structural isomers of corticosterone by performing MS3 analysis with an ion trap.