Nanostructure-initiator mass spectrometry (NIMS) imaging using soft laser desorption/ionization has proven to be a powerful tool in localizing the spatial distribution of intact biomolecules. NIMS specifically has been demonstrated to have high sensitivity and low background, particularly in the low mass range <1,000 Da, making this technique well suited for metabolic imaging studies. Here, we describe NIMS imaging for direct analysis of metabolite composition across a sectioned biospecimen.