Hydroxypyridinones (HOPOs) form outstanding building blocks for the development of a variety of agents in the field of metal chelation. The pyridinone ring is easily synthesized and readily converted into tetradentate, hexadentate, and octadentate chelators. There is considerable potential for the control of the stereochemistry of the resulting metal complex and hence the properties of these multidentate molecules. Their ability to rapidly bind hard metals in aqueous media has facilitated the development of efficient applications in both biological and medical contexts. In this Review, an in-depth analysis of the synthetic methodologies for HOPO-based ligands is presented, as well as the many aspects to achieve optimal biological activity. Recent advances and current challenges for the future application of HOPO structures are outlined. The present flourishing development of drug candidates and diagnostic agents based on this chemical scaffold opens access to many new applications in analytical, environmental, and clinical science.