Chemiluminescence is a fascinating phenomenon that evolved in nature and has been harnessed by chemists in diverse ways to improve life. This Account tells the story of our research group's efforts to formulate and manifest spiroadamantane 1,2-dioxetanes with triggerable chemiluminescence for imaging and monitoring important reactive analytes in living cells, animals, and human clinical samples. Analytes like reactive sulfur, oxygen and nitrogen species, as well as pH and hypoxia can be indicators of cellular function or dysfunction and are often implicated in the causes and effects of disease. We begin with a foundation in binding-based and activity-based fluorescence imaging that has provided transformative tools for understanding biological systems. The intense light sources required for fluorescence excitation, however, introduce autofluorescence and light scattering that reduces sensitivity and complicates in vivo imaging. Our work and the work of our collaborators were the first to demonstrate that spiroadamantane 1,2-dioxetanes had sufficient brightness and biological compatibility for in vivo imaging of enzyme activity and reactive analytes like hydrogen sulfide (H2S) inside of living mice. This launched an era of renewed interest in 1,2-dioxetanes that has resulted in a plethora of new chemiluminescence imaging agents developed by groups around the world. Our own research group focused its efforts on reactive sulfur, oxygen, and nitrogen species, pH, and hypoxia, resulting in a large family of bright chemiluminescent 1,2-dioxetanes validated for cell monitoring and in vivo imaging. These chemiluminescent probes feature low background and high sensitivity that have been proven quite useful for studying signaling, for example, the generation of peroxynitrite (ONOO-) in cellular models of immune function and phagocytosis. This high sensitivity has also enabled real-time quantitative reporting of oxygen-dependent enzyme activity and hypoxia in living cells and tumor xenograft models. We reported some of the first ratiometric chemiluminescent 1,2-dioxetane systems for imaging pH and have introduced a powerful kinetics-based approach for quantification of reactive species like azanone (nitroxyl, HNO) and enzyme activity in living cells. These tools have been applied to untangle complex signaling pathways of peroxynitrite production in radiation therapy and as substrates in a split esterase system to provide an enzyme/substrate pair to rival luciferase/luciferin. Furthermore, we have pushed chemiluminescence toward commercialization and clinical translation by demonstrating the ability to monitor airway hydrogen peroxide in the exhaled breath of asthma patients using transiently produced chemiluminescent 1,2-dioxetanedione intermediates. This body of work shows the powerful possibilities that can emerge when working at the interface of light and chemistry, and we hope that it will inspire future scientists to seek out ever brighter and more illuminating ideas.