Imaging small polar metabolites and analyzing their in vivo dynamics with stable isotope-labeled (SIL) tracing through various biochemical pathways, including the citric acid (TCA) cycle, glycolysis, and amino acid metabolism, have gained substantial interest over the years. However, imaging these small polar metabolites across different tissue types is limited due to their lower ionization efficiencies and ion suppression from larger abundant biomolecules. These challenges can be further exacerbated with SIL studies, which require improvements in sample preparation and method sensitivity. Solvent pretreatments before matrix application on a tissue section have the potential to improve the sensitivity of metabolite imaging; however, they are not yet widely optimized across tissue types. Furthermore, there is a recurring concern about metabolite delocalization from such wash treatments that require “spatial validation”. Here, we optimized a simple “basic hexane” wash method that improved sensitivity up to several folds for a broad range of polar and 2H-labeled metabolites across five different mouse organ tissues (kidney, heart, brain, liver, and brown adipose tissue). Notably, we provided region-specific quantification of 51 metabolites using laser microdissection (LMD)-LC-MS/MS to validate their localization observed in MALDI-MSI analysis after the basic hexane wash. Overall, we reported an improved MALDI-MSI sample pretreatment method with a “spatial validation” workflow for sensitive and robust imaging of polar metabolite distributions in mouse organs.