A consequence of chromophores being excited at certain wavelengths is a release of several forms of energy, typically via fluorescence emission. In addition to fluorescence emission, excited chromophores may transfer energy to the surrounding molecules, including molecular oxygen, leading to the generation of reactive oxygen species (ROS). Chromophores that have been demonstrated to be effective in generating ROS are known as photosensitizers. This chapter discusses the application of photosensitizers in inactivating proteins in a spatiotemporal manner, denoted the chromophore light inactivation (CALI) method, and the development of CALI tools that have improved our understanding of protein functions and dynamics. Further, controlling ROS generation using highly effective photosensitizers has permitted optogenetically controlled cell ablation in living organisms and has applications in photodynamic therapy. In the future, it will be possible to activate concomitantly several colours of photosensitizers, with different excitation wavelengths, allowing the study of multiple proteins or cell functions, within a single cell or an organism.