Heightened concentrations of reactive metabolites, including reactive oxygen species (ROS), can damage all macromolecules leading to the erosion of cellular fidelity. In this regard, the control of ROS in the nuclues is essential for cellular homeostasis, and dysregulation of nuclear ROS has been attributed to multiple pathologies and the mechanism of action of certain chemotherapies. How nuclear ROS is generated, detoxified and sensed is poorly understood, and stems in part, from a historical lack of tools that allow for its precise generation and detection. Here, we summarize the latest advances in chemical biology inspired approaches that have been developed to study nuclear ROS and highlight how these tools have led to major breakthroughs in understanding its regulation. The continued development and application of chemical biology approaches to understand nuclear ROS promises to unlock fundamental insights into human physiology and disease.