Iron is crucial for fundamental metabolic processes in most living organisms. Iron deficiency is the most common cause of anemia and affects about one-third of the population of the world. Iron overload, which may develop due to excessive dietary iron uptake (e.g., iron loading anemias, hereditary hemochromatosis), or repeated blood transfusions (e.g., sickle cell disease, beta-thalassemia major), is also deleterious. Excess free iron causes tissue damage and organ failure due to the formation of reactive oxygen species. In addition, the pathogenicity of numerous infections is affected by the iron status of the human host. Iron homeostasis is thus a tightly regulated process that, in healthy individuals, ensures the delivery of a sufficient amount of iron to cells and tissues according to their requirements, preventing excessive accumulation of the metal that could promote infections or result in tissue injury. Our knowledge of the mechanisms that underlie cellular and systemic iron homeostasis has greatly increased over the last two decades. However, iron-related disorders remain a major global health issue worldwide. Current treatments are often ineffective and new therapies are needed. In addition, deregulated iron homeostasis is implicated in an increasing number of ageing-associated diseases, from cancer to neurodegenerative, inflammatory and metabolic disorders. The goal of this collection is to present standardized protocols as well as innovative methodological developments to researchers interested in this rapidly evolving field of research.