Nitric oxide (NO) produced in endothelial cells exerts important roles in the vascular system. In recent years, posttranslational modifications induced by NO have been increasingly studied and, among them, cysteine modification by S-nitrosylation (also called S-nitrosation) has been hypothesized to represent a relevant mechanism for cell signaling. Thus, knowledge of the proteins that can be S-nitrosylated in endothelial cells will help to better understand the possible role of this modification. We describe a protocol to identify the S-nitrosylome or S-nitrosoproteome of endothelial cells, based on the specific derivatization of the S-nitrosylation, substituting it by a biotinylation, and the purification of the biotinylated proteins.