High content imaging is a foundational technology platform in pre-clinical drug discovery, supporting the generation of key decision-making data at most, if not all, points along the journey from target identification to the development of candidate therapeutics. The breadth of available technology and the biology to which it is applied is vast, and recent advances in the ability to generate and interpret huge datasets are a paradigm shift in how this technology can be used on a large scale in early drug discovery. This comprehensive resource covers state-of-the-art technologies, essential supporting infrastructure and data analysis approaches to support pre-clinical drug discovery. Chapters will describe key considerations and recommendations for establishing an assay for high content imaging microscopy platforms, what is required to support the use of such platforms, critical image handling infrastructure, and also how to apply high content imaging. Additionally, case studies and examples will provide a guide to the latest advances in the field, such as cell painting/morphological profiling, the inclusion of complex cellular and more advanced 3-dimensional cell models, and the use of traditionally low throughput techniques to support hit and target discovery. High Content Imaging in Drug Discovery brings together the many advances, challenges, and their solutions into a single essential resource for scientists and leaders working in high content imaging and seeking to establish capability within their drug discovery processes.