Atherosclerosis is one of the most common causes of death in developed countries. Atherosclerosis is an inflammatory process that results in the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction and stroke. Although certain risk factors (dyslipidemias, diabetes, hypertension) and humoral markers of plaque vulnerability (C-reactive protein, interleukin-6, -10 and -18, CD-40L) have been identified, a highly sensitive and specific biomarker or protein profile, which could provide information on the stability/vulnerability of atherosclerotic lesions, remains to be identified. Recently, we have described a novel strategy consisting in the proteomic analysis of proteins released by normal and atherosclerotic arterial walls in culture. This method enables harvesting of proteins that are only secreted by pathological or normal arterial walls. By focusing only on the secreted proteins found in the tissue culture media, there is an intended bias toward those molecules that would have a higher probability of later being found in plasma. Using this approach, we have shown that carotid atherosclerotic plaques cultured in vitro are able to secrete proteins, and also that a differential pattern of protein secretion of normal arteries vs pathological ones has been observed. In this chapter, the proteomic analysis of the human atheroma plaque secretome is described.