With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT₂-receptor [AT₂R] as potential new drug targets. While it is known that AT₂R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT₂R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT₂R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT₂R ligands, primarily for distinguishing between AT₂R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT₂R-transfected CHO cells is a reliable assay for the characterization of AT₂R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT₂R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT₂R-transfected HEK-293 cells are unresponsive to AT₂R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT₂R antagonists, are partial agonists at the AT₂R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT₂R-CHO cells that is suitable for the characterisation of novel and established AT₂R ligands.