Abstract Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells. GSH maintains cellular redox homeostasis and is involved in numerous biological processes. These important functions are dynamically regulated by the intracellular concentration and distribution of GSH. Currently, the concentration of intracellular GSH is derived from either cell lysates or GSH-S-transferase (GST) dependent probes. These approaches, however, cannot provide information about the real-time dynamics of GSH concentration changes. We reported the first reversible reaction-based GSH probe (ThiolQuant Green) that can perform single-point quantification of GSH levels in living cells and developed the 2nd generation GSH probe—RealThiol (RT)—that allows real-time monitoring of GSH dynamics in living cells. We filed a patent on these GSH probes (WO2016025382) and have shared the RealThiol probe with >70 redox biology labs around the world. Additionally, the Division of Cancer Treatment & Diagnosis Laboratory Support at National Cancer Institute (NCI) is interested in applying our GSH probes in their drug screening assays and requested gram quantity of RT probes. The overarching goal for this Phase I project is to develop probes that can monitor the dynamics of the concentration change of GSH in real- time with subcellular specificity and to provide a complete toolset to study GSH-related redox biology. For Phase II commercialization, we will focus on both basic and clinical research. We will develop assay kits for GSH-related biology, such as ferroptosis. We will also apply our GSH probes in clinical samples to help identify patients with drug/radiation-resistant tumors.