Chagas disease is an infectious disease caused by the parasite Trypanosoma cruzi, with heart failure being a severe complication of its chronic stage. T. cruzi has three lifecycle stages: epimastigote, trypomastigote, and amastigote. Amastigotes are the intracellular form that invade mammalian host cells, replicate in their cytoplasm, and cause tissue damage. Due to the small size of T. cruzi amastigotes, fine spatial resolution would be beneficial. Current metabolomics research on Chagas disease primarily focuses on studying metabolites through widely used bulk analysis techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, conducting comprehensive molecular studies of Chagas disease is constrained by the inherent limitations, including sample loss and lack of spatial resolution, of these traditional analytical tools. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is an advanced analytical method that combines ambient sampling and ionization with mass spectrometry to produce spatially resolved chemical maps of sample surfaces. This chapter describes the general procedures of using DESI-MSI techniques to study metabolic alterations in mouse hearts infected with T. cruzi, with high spatial resolution, preserving tissue integrity and offering new insights into potential therapeutic targets.