Nitric Oxide (NO center dot) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO center dot in vivo and in vitro, the real-time monitoring of NO center dot at the single-cell level is very challenging. The physiological or pathological effects of NO center dot are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO center dot are highly desirable. Recently, we expanded the pallet of NO center dot indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO center dot) probes (geNOps) that directly respond to cellular NO center dot fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO center dot signals in response to two different chemical NO center dot liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO center dot levels as compared with the inorganic NO center dot donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+ -dependent NO center dot formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO center dot signals in diverse experimental setups.