Vascular endothelium has attracted extensive attentions due to its important role in many physiological and pathological processes. Many methods have been developed to study the components and their functions in vascular endothelium. Here we reported a novel approach to investigate vascular endothelium using normal rat lungs as the model. We perfused lung vascular beds with Sulfo-NHS-LC-Biotin to label endothelial membrane proteins. The biotinylated proteins were isolated from lung homogenate using immobilized monomeric avidin and confirmed to be highly pure endothelial membrane proteins with little contamination of intracellular proteins. These biotinylated proteins were used as immunogens for development of monoclonal antibodies. Indeed, newly generated monoclonal antibodies have revealed different expression patterns of proteins across tissues using immunoblotting and immunohistochemistry. Some proteins were found highly specifically expressed to capillary vessels of pulmonary vasculature. As being explored in the study, this method has also been proven useful for investigating vasculature of other organs.