Over the past decades, research has made impressive breakthroughs towards drug delivery systems, resulting in a wide range of multifunctional engineered nanoparticles with biomedical applications such as cancer therapy. Despite these significant advances, well-designed nanoparticles rarely reach the clinical stage. Promising results obtained in standard 2D cell culture systems often turn into disappointing outcomes in in vivo models. Although the overall majority of in vitro nanoparticle research is still performed on 2D monolayer cultures, more and more researchers started acknowledging the importance of using 3D cell culture systems, as better models for mimicking the in vivo tumor physiology. In this review, we provide a comprehensive overview of the 3D cancer cell models currently available. We highlight their potential as a platform for drug delivery studies and pinpoint the challenges associated with their use. We discuss in which way each 3D model mimics the in vivo tumor physiology, how they can or have been used in nanomedicine research and to what extent the results obtained so far affect the progress of nanomedicine development. It is of note that the global scientific output associated with 3D models is limited, showing that the use of these systems in nanomedicine investigation is still highly challenging.