For a patient with metastatic colorectal cancer, there are limited clinical options aside from chemotherapy. Unfortunately, the development of new chemotherapies is a long and costly process. Aside from ethical concerns, typical in vivo research on these toxic chemotherapeutic drugs is low throughput, time consuming and costly. In this project, two novel in vitro platforms are employed to better understand the effects of chemotherapeutic treatments on colon cancer cells: tumor spheroids and paper-based cultures. The first part of this thesis utilizes 3-dimensional cell culture tumor spheroids. These multi-cellular aggregates, or spheroids, of colorectal cancer cells have been shown to exhibit many of the physiological and chemical gradients of a typical clinical tumor, while still maintaining the flexibility of a cell culture system. This project utilizes a 3D printed fluidic device that facilitates high-throughput treatment of these tumor spheroids across a semipermeable membrane. The device also allows for a dynamic drug-dosing gradient similar to a typical in vivo pharmacokinetic profile. The treated tumor spheroids are evaluated with MALDI-Imaging Mass Spectrometry to elucidate the spatial  distribution of the drug molecules and corresponding metabolites. Spheroids are also analyzed for the quantitative proteomic changes induced by treatment. This innovative system enables both pharmacokinetic and pharmacodynamic evaluations for a wide range of compounds, and can have a transformative impact on the pre-clinical evaluation of drug candidates. The second part of this thesis focuses on another a novel in vitro platform, paper- based cultures (PBCs). Individual layers of paper are seeded with colon cancer cells, stacked together into a 3D structure containing a diffusion-limited environment generated with the use of diffusion limiting membranes and stainless steel holders. These PBCs display monotonic gradients similar to the gradients found in poorly vascularized tumors, making paper-based cultures a 3D tumor mimic. Individual layers of PBCs were characterized for cell viability, proliferation and drug response. PBCs were dosed with chemotherapeutics and monitored for the cellular response at different regions within the PBCs. We also developed a methodology to perform MALDI-Imaging Mass Spectrometry on PBCs. The described analytical platforms enable preclinical chemotherapeutic analysis and are readily available for the exploration of new and creative cancer treatments.