Cancer metabolic variability has a significant impact on both diagnosis and treatment outcomes. The discovery of novel biological indicators and metabolic dysregulation, can significantly rely on comprehension of the modified metabolism in cancer, is a research focus. Tissue histology is a critical feature in the diagnostic testing of many ailments, such as cancer. To assess the surgical margin of the tumour on patients, frozen section histology is a tedious, laborious, and typically arbitrary method. Concurrent monitoring of ion images in tissues facilitated by the latest advancements in mass spectrometry imaging (MSI) is far more efficient than optical tissue image analysis utilized in conventional histopathology examination. This article focuses on the “desorption electrospray ionization (DESI)-MSI” technique’s most recent advancements and uses in cancer research. DESI-MSI can provide wealthy information based on the variances in metabolites and lipids in normal and cancerous tissues by acquiring ion images of the lipid and metabolite variances on biopsy samples. As opposed to a systematic review, this article offers a synopsis of the most widely employed cutting-edge DESI-MSI techniques in cancer research.