Protein biomarker discovery in blood plasma and serum is severely hampered by the vast dynamic range of the proteome. With protein concentrations spanning 12 orders of magnitude, conventional mass spectrometric analysis allows for detection of only a few low-abundance proteins. Prior depletion of high-abundant proteins from the sample can increase analytical depth considerably and has become a widely used practice. We describe in detail an affinity depletion method that selectively removes 14 of the most abundant proteins in plasma and serum.