Glutathione (GSH) plays a crucial role in human pathologies. Near-infrared fluorescence-based sensors capable of detecting intracellular GSH in vivo would be useful tools to understand the mechanisms of diseases. In this work, two cyanine-based fluorescent probes, 1 and 2, containing sulfonamide groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzenesulfonamide group, shows that it is cell-membrane-permeable and can selectively detect thiols such as GSH, cysteine (Cys), and homocysteine (Hcy) in living cells. The response of 1 to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular levels of GSH modualated by H2O2. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.