Reactive oxygen species (ROS), such as hydrogen peroxide, are important products of oxygen metabolism that, when misregulated, can accumulate and cause oxidative stress inside cells. Accordingly, organisms have evolved molecular systems, including antioxidant metalloenzymes (such as superoxide dismutase and catalase) and an array of thiol-based redox couples, to neutralize this threat to the cell when it occurs. On the other hand, emerging evidence shows that the controlled generation of ROS, particularly H(2)O(2), is necessary to maintain cellular fitness. The identification of NADPH oxidase enzymes, which generate specific ROS and reside in virtually all cell types throughout the body, is a prime example. Indeed, a growing body of work shows that H(2)O(2) and other ROS have essential functions in healthy physiological signaling pathways. The signal-stress dichotomy of H(2)O(2) serves as a source of motivation for disentangling its beneficial from its detrimental effects on living systems. Molecular imaging of this oxygen metabolite with reaction-based probes is a powerful approach for real-time, noninvasive monitoring of H(2)O(2) chemistry in biological specimens, but two key challenges to studying H(2)O(2) in this way are chemoselectivity and bioorthogonality of probe molecules. Chemoselectivity is problematic because traditional methods for ROS detection suffer from nonspecific reactivity with other ROS. Moreover, some methods require enzymatic additives not compatible with live-cell or live-animal specimens. Additionally, bioorthogonality requires that the reactions must not compete with or disturb intrinsic cellular chemistry; this requirement is particularly critical with thiol- or metal-based couples mediating the major redox events within the cell. Chemoselective bioorthogonal reactions, such as alkyne-azide cycloadditions and related click reactions, the Staudinger-Bertozzi ligation, and the transformations used in various reaction-based molecular probes, have found widespread application in the modification, labeling, and detection of biological molecules and processes. In this Account, we summarize H(2)O(2) studies from our laboratory using the H(2)O(2)-mediated oxidation of aryl boronates to phenols as a bioorthogonal approach to detect fluxes of this important ROS in living systems. We have installed this versatile switch onto organic and inorganic scaffolds to serve as "turn-on" probes for visible and near-infrared (NIR) fluorescence, ratiometric fluorescence, time-gated lanthanide luminescence, and in vivo bioluminescence detection of H(2)O(2) in living cells and animals. Further chemical and genetic manipulations target these probes to specific organelles and other subcellular locales and can also allow them to be trapped intracellularly, enhancing their sensitivity. These novel chemical tools have revealed fundamental new biological insights into the production, localization, trafficking, and in vivo roles of H(2)O(2) in a wide variety of living systems, including immune, cancer, stem, and neural cell models.