Understanding the biological and dose-related factors that influence the susceptibility of tumors to photodynamic therapy (PDT) and PDT-based combinations requires advancements in many complementary areas. Among these includes the development of 3D cancer models that, in part, bridge the gap between monolayer cultures and complex biology in vivo. Unraveling the relative contributions of tumor architecture and microenvironmental cues—such as the communication of tumor cells with the surrounding matrix and heterotypic stromal partners—is an important part of a multifaceted discovery framework for designing and optimizing targeted strategies involving PDT. This chapter describes the value, challenges and opportunities associated with using 3D models in PDT research, with a particular focus on the insights provided by non-adherent multicellular spheroid cultures, along with more recent studies in adherent 3D models. The utility of these models for investigating dose-related parameters, including photosensitizer distribution, irradiance and energy density, along with the importance of developing a suitable analysis framework for characterizing treatment responses and designing combination regimens, is discussed. A perspective on the opportunities provided by increasingly sophisticated bioengineered 3D systems that integrate concepts and techniques from an array of research areas, including tissue engineering, cancer biology and microelectromechanical systems, for designing PDT-based regimens that overcome tumor heterogeneity and treatment resistance is also provided.