Cancer therapeutics have the potential to be lifesaving, and healthcare practitioners utilize a comprehensive methodology to make well-considered decisions regarding the most suitable clinical interventions for individuals with cancer. Despite advancements, persistent clinical gaps remain, especially in precisely determining the optimal personalized therapy for each patient. In retrospective studies employing Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) with formalin-fixed, paraffin-embedded (FFPE) samples from gynecological cancers, we have successfully predicted responses to chemotherapy in Ovarian Cancer and identified metastasis in Endometrial Cancer based on the proteomic profile of the primary tumor. This success is attributed to the refinement of sample preparation, workflows, and data analysis techniques specific to MALDI-MSI. Additionally, we have utilized MALDI-MSI to monitor drug penetration in primary Ovarian Cancer spheroids and evaluate the response to traditional chemotherapy.