S-nitrosoglutathione reductase (GSNOR) is a multifunctional enzyme. It can catalyze NADH-dependent reduction of S-nitrosoglutathione (GSNO); as well as NAD+-dependent oxidation of hydroxymethylglutathione (HMGSH; an adduct formed by the spontaneous reaction between formaldehyde and glutathione). While initially recognized as the enzyme that is involved in formaldehyde detoxification, increasing amount of evidence has shown that GSNOR also plays a significant role in nitric oxide mediated signaling through its modulation of protein S-nitrosothiol signaling. In humans, GSNOR/S-nitrosothiols have been implicated in the etiology of several diseases including lung cancer, cystic fibrosis, asthma, pulmonary hypertension, and neuronal dysfunction. Currently, it is not possible to monitor the activity of GSNOR in live cells. In this article, we present a new compound, O-aminobenzoyl-S-nitrosoglutathione (OAbz-GSNO), which acts as a fluorogenic pseudo-substrate for GSNOR with an estimated Km value of 320 μM. The weak OAbz-GSNO fluorescence increases by approximately 14 fold upon reduction of its S-NO moiety. In live cell imaging studies, OAbz-GSNO is readily taken up by primary pulmonary endothelial cells and localizes to the same perinuclear region as GSNOR. The perinuclear OAbz-GSNO fluorescence increases in a time dependent manner and this increase in fluorescence is abolished by siRNA knockdown of GSNOR or by treatment with GSNOR-specific inhibitors N6022 and C3. Taken together, these data demonstrate that OAbz-GSNO can be used as a tool to monitor the activity of GSNOR in live cells.