Abstract: The development and evaluation of anticancer drugs face multifaceted challenges, including heterogeneity in drug biodistribution, complex pharmacokinetic properties, and difficulties in deciphering drug resistance mechanisms. While traditional analytical methods enable in vitro quantification of total drug concentrations, they fail to elucidate the spatiotemporal dynamics of drugs within tissue microenvironments. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), a label-free molecular imaging technology characterized by high sensitivity and spatial resolution, overcomes these limitations by integrating mass spectrometry analysis with spatial information visualization. Its core strength lies in the simultaneous in situ detection and absolute quantification of drugs and their metabolites, providing a molecular-level precision tool for investigating tumor-targeted drug delivery efficiency, tissue penetration depth, and metabolic clearance pathways. In recent years, advancements in quantitative methodologies and calibration strategies have significantly enhanced the reproducibility and dynamic monitoring capabilities of quantitative mass spectrometry imaging (q-MSI). These innovations have demonstrated the unique value of MALDI-MSI in diverse applications, such as evaluating peritoneal permeation of paclitaxel, identifying irinotecan-resistant subpopulations, and tracking hepatic metabolism of doxorubicin. This review systematically outlines the technical principles and standardized workflows of MALDI-MSI, highlights its emerging applications in the development of antitumor nanomedicines, and underscores its distinctive advantages and broad prospects in advancing cancer therapeutics research.