Drug penetration into solid tumors is critical for the effectiveness of clinical chemotherapy. Failing to consider the efficiency of drug penetration can lead to fatal recurrence in many cancers. Three-dimensional (3D) cell cultures have served as an important model system and have contributed to valuable assays in drug discovery studies. However, limited methodologies result in incomplete evaluation of the distribution of many anticancer drugs and their metabolites. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging(MSI) is a powerful label-free technique for the investigation of the spatial distribution of molecules at complex surfaces. In this work, I demonstrate the application of MALDI-MSI in colon carcinoma multicellular tumor spheroids (MCTS) to assess the distribution of the anticancer drugs, irinotecan or platinum (Pt)-drugs as well as their metabolites. Nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) or ultra-performance liquid chromatography-tandem mass spectrometry in multiple reactions monitoring (MRM) mode (UPLC-MRM MS) combined with serial trysinization was also developed to validate and quantify drugs and metabolites in different regions of MCTS. These novel approaches allow the measurement of drug penetration and distribution in 3D culture mimics and provide a more cost and time-effective approach for the testing of new pharmaceuticals compared to animal models.