The study of cancer biology is replete with debate and disagreement over the various analytical stratagems ( in vitro vs. in vivo , animal vs. human) employed to develop therapeutic treatments. Yet, due to the highly variable nature of cancer cells and their acute sensitivity to extracellular microenvironments and culture conditions, it is critical that a culture model be chosen that closely resembles actual in vivo conditions in support of clinical relevance. As such, we hypothesized that the development of a three-dimensional (3D), tissue engineered human prostate cancer model would provide for a viable alternative, bridging the gap between issues with animal and in vitro models. In this study, we developed and tested the feasibility of such a model as well as a series of modified cell and molecular assays utilized to assess the effects of a given treatment on cell response. Human prostate cancer cells (PC3) were suspended in collagen matrices and variables such as gel thickness (1–3 mm), collagen density (0.5, 1, and 2 mg/mL), cell seeding density (1, 2.5, and 5 × 10 6 cells/mL), and top-seeded vs. 3D-seeded, were evaluated for their influence on cell survival, proliferation, and response to a therapeutic stress. With the engineered model we were able to assess, using modified techniques, membrane integrity, metabolic function, and various cell death mechanisms. From our evaluations it was found that PC3 cells were able to survive and proliferate under several different model parameters, but a 2 mm thick collagen gel at 2 mg/mL seeded with 5 × 10 6 cells/mL was chosen as the standard configuration. Additionally, the application of cryosurgical therapy to our 3D model revealed cell behavior characteristics similar to that of in vitro monolayer data. Through this study, we established a 3D, tissue engineered in vivo -like prostate cancer model along with a series of assessment techniques for utilizations in cancer research studies. Using this model, our aim is to better understand cancer cell responses to low temperatures, thus providing a new avenue for the improvement of cryosurgery.