Nitric oxide (NO) is a key signalling molecule in the vascular endothelium. It can induce different post-translational modifications in mammalian proteins that can alter their functionality, among which incorporation of a NO group in cysteine thiols, called S-nitrosation or S-nitrosylation, is one of the best characterized. Identification of the proteins that are susceptible to this modification would help us to determine the relevance of this modification in physiological and pathophysiological conditions. For this purpose, we have used a proteomic approach to identify S-nitrosylated proteins in endothelial cells, which includes replacing S-nitrosylation by a specific biotinylation and subsequent purification (called the "biotin switch" method). We have applied this methodology to identify proteins that are S-nitrosylated in endothelial cells acutely exposed to S-nitroso-l-cysteine, a physiological S-nitrosothiol. We describe the identified proteins and discuss the characterization of their S-nitrosylation, confirming the validity of the methodology for approaching the description of the "nitrosylome."