One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Between different proteomics studies, the secretome is a valuable tool in the search for biomarkers locally released by a studied tissue and remains particularly important while working with vascular tissues, since the secreted proteins would be probably shed to the blood. In this chapter, we described a method to validate the origin of secreted proteins in human aortic valves, demonstrating their synthesis and release by the tissue and ruling out blood origin.