This review surveys recent advances in optical spectral detection of reactive oxygen species (ROS), particularly singlet oxygen, superoxide, hydroxyl radical, and hydrogen peroxide. Advances using nanoparticles and self-organizing nanostructures as well as optical detection schemes are included. Measurements using plasmonic, luminescent, photocatalytic, or self-organizing nanoparticles are highlighted. The large number of spectrophotometric and luminescent probe methods are categorized by ROS sensing mechanism, signaling mode, (de)activation mechanism, if any, and spectral chromaticity. Reports describing multicomponent ROS detection or novel nanoscale probes are discussed. Measurements using ratiometric, multichannel, or time-resolved detection and nonlinear spectral transitions are reviewed. The focus on developing probe molecules for spectral detection documented over the last 20 years has continued, with sustained emphasis on luminescence detection, but with less focus on spectrophotometric measurements. Use of nanoparticles as probes, probe carriers, and compartmentalization agents in ROS detection is increasing. On the other hand, incorporation of advanced spectral methods, such as nonlinear transition and multichannel detection, is increasing slowly in ROS analysis. This indicates there is a substantial opportunity to develop ROS measurements with use of a synergistic combination of (multi)functional nanoscale systems and advanced optical detection methods to optimize the detection limit, selectivity, and response time.