Reactive oxygen species (ROS) are continuously produced, transformed, and consumed in all living organisms due to aerobic life. Traditionally, ROS are viewed as causing oxidative stress and damage, leading to tissue and organ decline in aging and disease. However, emerging data suggest that ROS can also contribute to physiology and increased fitness under certain conditions. This perspective discusses the factors that lead ROS to become signaling or stress agents, highlighting how advancements in understanding the chemistry of ROS can advance our understanding of their diverse contributions to biology. The development of new tools to study these small molecules and their reactivity in complex biological systems is an important aspect of this emerging field. ROS can modify biomolecules through oxidation-reduction reactions, and their chemical reactivity is influenced by their identity, concentration, and local environment. The article covers the generation of ROS in various cellular compartments, such as mitochondria, endoplasmic reticulum, and cell membranes, and their roles in processes like protein folding, cell migration, circadian rhythm, and stem cell proliferation. It also discusses the control mechanisms that cells use to regulate ROS chemistry for signaling functions, including colocalization of ROS sources and targets, modulation of local redox buffer capacity, and membrane transport and sequestration. The article concludes by emphasizing the importance of chemical biology approaches in elucidating the complex redox processes in biological systems and the need for new methods to monitor ROS in living systems.Reactive oxygen species (ROS) are continuously produced, transformed, and consumed in all living organisms due to aerobic life. Traditionally, ROS are viewed as causing oxidative stress and damage, leading to tissue and organ decline in aging and disease. However, emerging data suggest that ROS can also contribute to physiology and increased fitness under certain conditions. This perspective discusses the factors that lead ROS to become signaling or stress agents, highlighting how advancements in understanding the chemistry of ROS can advance our understanding of their diverse contributions to biology. The development of new tools to study these small molecules and their reactivity in complex biological systems is an important aspect of this emerging field. ROS can modify biomolecules through oxidation-reduction reactions, and their chemical reactivity is influenced by their identity, concentration, and local environment. The article covers the generation of ROS in various cellular compartments, such as mitochondria, endoplasmic reticulum, and cell membranes, and their roles in processes like protein folding, cell migration, circadian rhythm, and stem cell proliferation. It also discusses the control mechanisms that cells use to regulate ROS chemistry for signaling functions, including colocalization of ROS sources and targets, modulation of local redox buffer capacity, and membrane transport and sequestration. The article concludes by emphasizing the importance of chemical biology approaches in elucidating the complex redox processes in biological systems and the need for new methods to monitor ROS in living systems.