The tumor microenvironment (TME) is composed of cancer, immune, and stromal cells that interact through cell-to-cell contact and a diverse milieu of cytokines, chemokines, growth factors, and proteases. Several reports have linked the presence of specific cell subtypes with tumor stages, prognosis, and patient survival. Understanding cancer cell behavior and their response to treatment within the tumor microenvironment is essential to prevent establishment, growth, and progression of tumors. Many synthetic and biological agents have been tested using cell-based assays, which do not provide reliable predictive capacity for drug candidate performance in vivo. In this chapter, we discuss about the benefits of an air pouch tumor model, in which tumor cells are inoculated inside an air pouch created on the back of the mouse. The air pouch cancer model serves as a confined/localized tumor microenvironment, where direct contact of drug candidates and tumor cells is guaranteed in a tumor microenvironment context. Therefore, the efficacy of the therapeutic agent can be accurately assessed in vivo.