The endothelium is more than just a passive interface between the blood and the vessel wall. Since the pioneering discovery of nitric oxide as an important endothelium-derived vasorelaxing molecule three decades ago, vascular research has developed exponentially and remains fascinating for the entire research community. Endothelial dysfunction is a pathological condition characterized by an imbalance between vasodilatating and vasoconstricting substances. Most, if not all, cardiovascular risk factors have been attributed with endothelial dysfunction and their therapeutic modification with an improvement in vascular function. This overview aims to provide a glimpse into this fascinating research field with the emphasis on vasoactive substances and the assessment of endothelial function.