The field of biochemistry and pharmacology of nitric oxide (NO) has various clinical perspectives, such as cardiovascular and metabolic disorders; neurovascular and neurodegenerative diseases; muscular–skeletal disorders; ocular, respiratory, and a large series of inflammation-related pathologies; and, on top of these, infective and neoplastic diseases. In all these disorders, vascular endothelial cells (ECs) play a crucial role as both NO producers and NO responders. Depending on the pathology and pathophysiological mechanisms, different pharmacological approaches can be developed and used, either to stimulate NO synthases (NOS), improve NO availability, and activate downstream NO-related pathways or, on the opposite, to downregulate NOS, scavenge NO, and inhibit NO-related signaling. Introducing endothelial cell cultures, genetic manipulation, and molecular studies allows us to thoroughly investigate the role of NO and its signaling in EC functional responses and angiogenesis. The present chapter presents the cell types and the cellular assays used to study the functional aspects of NO-related strategies in vascular endothelium. By using selective biochemical inhibitors of signaling pathways, the involvement of intracellular messengers can be assessed and verified in functional responses associated with EC proliferation, migration, and permeability.