Colorectal cancer (CRC) represents an increasingly large threat as it is estimated that 8% of cancer-related deaths worldwide are due to advanced forms of CRC.When detected in early stages, CRC has a very good prognosis upon resection of the primary tumor and continued monitoring of the colon for other aberrant growths.CRC's biggest threat lies in its ability to readily metastasize to the liver, lymph system and lungs when undetected.Identifying interactions between distinct populations of cells in the tumor architecture that promote metastasis and evade therapy represents a key step in combating this disease. Using matrix-assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS), imaging mass spectrometry (IMS) offers the ability to preserve important spatial information in the X,Y and Z planes to observe any analyte detected over a broad mass range in its native spatial distribution within a sample. Although initially applied to thin sections of mammalian tissues, the application of IMS to sections of three dimensional cell culture tumor mimics has led to a dramatic increase in the ability to study and understand the complex interactions between tumor microenvironments. The experimental flexibility inherent to cell culture models combined with the robustness of IMS allows for the study of protein expression patterns across the structure of the spheroid, siRNA-mediated knockdown of specific proteins , drug penetration and efficacy studies and lipid expression and localization analysis. The application of chemometrics to IMS datasets has greatly enhanced our understanding of these complex experiments. Overall, these studies have evolved the application of IMS to three dimensional cell culture models into a robust and viable experimental technique that will have an enormous impact on the understanding of the complex interaction between tumor microenvironments and how drug treatment and chemotherapeutics affect tumor microenvironments.