Nitric oxide (NO) is a highly reactive and freely diffusible molecule that has been implicated in regulating a diverse range of physiological processes from synaptic plasticity to vasodilation. NO elicits these effects through different signalling pathways, which are largely dependent on the concentration of NO produced. At low and physiological concentrations, NO generation from nitric oxide synthase (predominantly neuronal or endothelial nitric oxide synthase) leads to the activation of its target receptor soluble guanylyl cyclase and subsequent generation of cyclic guanosine monophosphate which leads to the activation of a wide range of downstream signalling molecules such as protein kinases. At higher concentrations, such as those produced in response to pathophysiological conditions, NO release can result in post-translational modifications. These include S-nitrosylation of cysteine residues and nitration of tyrosine residues, both of which alter protein function, usually detrimentally. The study of NO signalling usually involves application of exogenous NO via various donors. However the use of NO donors in research can be problematic due to the unknown release capacity of each donor. This has led to countless publications of contradictory findings due to the use of different donors and concentrations across studies. In order to better characterise the release profile of NO from commonly used NO donors, we measured temporal release profiles following varying storage times at 4oC and -20oC of different donors at multiple concentrations. NO release was detected in standard phosphate buffered saline over time using NO sensing electrodes. The NO microsensor chosen for this study (NOPF100; World Precision Instruments Ltd) possess a multi-layered selective coating that eradicates non-specific detection providing reliable NO measurements. We found that diazeniumdiolate donors such as NOC-5 and PAPA NONOate initially release high levels of NO but decay substantially within days, whereas S-nitrosothiols SNP and GSNO stocks show greater stability in solution releasing consistent and lower levels of NO over several days. Furthermore, in all donors tested, the amount of released NO differs between long-term frozen and fresh stock solutions. Therefore our data provides a systematic and comprehensive comparison of NO release by different donors which provides insightful information for studying nitrergic signalling and allows a better evaluation of reported nitrergic signalling outcomes.