Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. In the present chapter, we detail the characterization and analysis of human arterial tissue secretome by two-dimensional electrophoresis (2-DE) and nano-liquid chromatography on-line coupled to mass spectrometry (nLC-MS/MS). General strategies shown here can be extended to other tissue secretome studies.