For decades, 2D cell culture format on plastic has been the main workhorse in cancer research. Though many important understandings of cancer cell biology were derived using this platform, it is not a fair representation of the in vivo scenario. In this review, both established and new 3D cell culture systems are discussed with specific references to anti-cancer drug and nanomedicine applications. 3D culture systems exploit more realistic spatial, biochemical and cellular heterogeneity parameters to bridge the experimental gap between in vivo and in vitro settings when studying the performance and efficacy of novel nanomedicine strategies to manage cancer. However, the complexities associated with 3D culture systems also necessitate greater technical expertise in handling and characterizing in order to arrive at meaningful experimental conclusions. Finally, we have also provided future perspectives where cutting edge 3D culture technologies may be combined with under-explored technologies to build better in vitro cancer platforms.