This comprehensive guide provides an introduction to High Content Imaging (HCI) and High Content Screening (HCS), outlining best practices for assay development. It serves as a foundational resource for new practitioners in the field, detailing state-of-the-art HCS approaches and discussing specific challenges associated with these techniques. The guidelines cover essential aspects such as image technologies and instruments used in HCS, assay concept and design, and the utility of HCS in various research areas. It explains how HCS, or automated microscope-based screening, measures biological activity in single cells or whole organisms following treatment with thousands of agents in multi-well plates. High Content Screening is widely used across the preclinical drug discovery pipeline, enabling the identification and validation of new drug targets or lead compounds, prediction of in vivo toxicity, and suggestion of pathways or molecular targets for orphan compounds. Beyond biological assays, HCS systems are also applied in chemistry and material science, for example, in screening crystallization conditions or assessing corrosion resistance. This digital resource is part of the Assay Guidance Manual, a widely recognized resource for developing assay formats compatible with High Throughput Screening (HTS) and Structure Activity Relationship (SAR) measurements. It provides detailed information on optimizing assay reagents, troubleshooting protocols, adapting assays for automation, and statistical validation of performance parameters.