4-(2-((2-hydroxybenzyl)amino)ethyl)-2,5-dimethoxybenzonitrile (25CN-NBOH) was first reported as a potent and selective serotonin 2A receptor (5-HT 2A R) agonist in 2014, and it has since found extensive use as a pharmacological tool in a variety of in vitro, ex vivo and in vivo studies. 25CN-NBOH is readily available from a synthetic perspective using standard chemical transformations, and displays favorable physiochemical properties in terms of stability and solubility. Due to its superior selectivity for 5-HT 2A R, 25CN-NBOH has been used to investigate the effects of selective 5-HT 2A R activation in vivo, and has thus become an important pharmacological tool for the exploration of 5-HT 2A R signaling in a range of animal models. In the present review, we outline the discovery of 25CN-NBOH, its pharmacological profile and major findings from studies where it has been used.