Strategies for removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low-abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high-abundance protein depletion process still represent common concerns. Here, we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system which is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed implementing this system, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels, but in a reproducible manner. The results suggest that multi-protein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies.