One important phase in drug development is to investigate distribution and delivery of therapeutic compounds to target organs identifying downstream associated metabolites. In tumors, one of the most important issues of pharmaceutical treatment is indeed the limited capability of chemotherapeutic drugs to penetrate tissues and reach all cells in a sufficient concentration inhibiting tumor growth. Mass-spectrometry imaging can be used to investigate the distribution of drugs, drug metabolites and metabolic indicators in the tumor microenvironment. However, to give pharmacologists a robust feedback on the efficacy of the different treatments, it is necessary to develop specific data analysis pipelines.