Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful analytical technique for detecting species with unpaired electrons, such as free radicals and transition metal complexes. This dissertation explores the development and application of EPR-based molecular probes to address key questions in redox biology and biomedical research. First, we synthesized and characterized a series of disulfide-dinitroxide probes for glutathione (GSH), a critical cellular antioxidant. These probes enable quantification of intracellular GSH levels based on EPR spectral changes following disulfide cleavage by GSH. Next, we developed an EPR-based method to measure superoxide (O2•–) production in red blood cells (RBCs), using the “spin trap”, CMH, that reacts with O2•– to yield a stable nitroxide radical, which has a robust EPR signal. This method was applied to study oxidative stress in diverse RBC contexts, including RBCs from sickle-cell disease (SCD) patients and healthy donors, transgenic mouse models of SCD, and transgenic mouse models of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Additionally, we designed a peptide-based EPR probe to detect matrix metalloproteinase-7 (MMP7) activity, providing proof-of-concept as a tool for monitoring MMP activity in disease contexts such as cancer. Collectively, this work advances the utility of EPR spectroscopy for probing oxidative stress and redox biology in living systems, with potential applications in diagnostics, drug screening, and disease monitoring.