Significance: Reactive Oxygen Species (ROS) may regulate signaling, ion channels, transcription factors and biosynthetic processes. ROS-related diseases can be either due to a shortage or to an excess of ROS. Recent advances: Since biological activity is not only achieved through ROS concentration but also by their spatial and temporal distribution, real-time imaging of ROS, possibly “in vivo”, has become a needful tool for scientists with potential for clinical translation. New imaging techniques, as well as new contrast agents in clinically established modalities, have been developed in the last decade. Critical issues: An ideal imaging technique should determine ROS changes with a high spatio-temporal resolution, detect physiologically relevant variations in ROS concentration and provide specificity towards different redox couples. Furthermore, for in vivo applications, bioavailability of sensors, tissue penetrance and a high signal to noise ratio are additional requirements to be satisfied. Future directions: None of here presented techniques fulfills all the requirements for clinical translation. The obvious way forward is to incorporate anatomical and functional imaging into a common hybrid imaging platform.