Histopathological assessment of tissue sections remains the gold standard technique for diagnosing cancer and is of utmost significance in analyzing cancer margins during surgeries. Given the challenges and time-consuming procedures required for frozen section histopathology during the intraoperative surgical margin assessment, there is a need for new technologies for rapid cancer detection based on diagnostic molecular signatures derived from the tissue. With recent developments in ambient ionization mass spectrometric imaging (MSI) techniques, a wide range of biomolecules can now be simultaneously visualized in the surgically resected specimen. This approach is considerably more effective and powerful than the conventional histopathology method of analyzing tissue samples based on a single optical image. This chapter aims to provide a comprehensive overview of the advancement of desorption electrospray ionization mass spectrometry imaging (DESI-MSI), an extremely valuable and emerging analytical technique that operates under ambient conditions without the need for any sample pretreatment. Tumor development is often marked by metabolic reprogramming, leading to distinct expression levels of molecular species in unhealthy and healthy tissues. DESI-MSI can intercept thousands of small molecules (metabolites, lipids, etc.) from a tissue specimen, rendering the spatial visualization of those species across the surface of the tissue. The increasing success of this technology in cancer margin evaluation, grading, and prognosis, along with ongoing advancements, makes DESI-MSI highly promising for future clinical implementation.