Integrating biological imaging into early stages of the drug discovery process can provide invaluable readouts of drug activity within complex disease settings, such as cancer. Iterating this approach from initial lead compound identification in vitro to proof-of-principle in vivo analysis represents a key challenge in the drug discovery field. By embracing more complex and informative models in drug discovery, imaging can improve the fidelity and statistical robustness of preclinical cancer studies. In this Review, we highlight how combining advanced imaging with three-dimensional systems and intravital mouse models can provide more informative and disease-relevant platforms for cancer drug discovery.