Microscopic imaging can enhance the drug discovery process by helping to describe how disease processes unfold and how potential therapies might intervene. Recently introduced technologies, and enhancements to existing techniques, are addressing technical issues that have limited the usefulness of microscopic imaging in the past. In particular, these innovations are improving spatial resolution, increasing tissue penetration, overcoming physical access issues and enhancing experimental throughput. Notable recent trends, which are discussed in this article, include the development of super-resolution microscopes, the incorporation of multiphoton techniques into intravital and fibre-optic microscopy and the automation of microscopy and image analysis for high-content screening. Together, these developments are augmenting the existing assays and disease models that are used in early drug discovery and, in some cases, enabling new ones.