Human biological fluids are complex matrices containing many elements (ions, lipids, sugars, proteins, etc.). Proteins are essential for biomarker and disease discovery though the most abundant proteins often provide very limited clinically relevant data, as can be the case for albumin or immunoglobulins. In this work, we focused on depleting albumin from human serum samples using an albumin depletion and low abundance protein enrichment kit, which enabled the detection of several low-abundance proteins. By specific software prediction (Ingenuity), enriched proteins known as biomarkers for various diseases were identified. By employing an optimized supplier’s protocol, loss of proteins was decreased and could be revealed by LC-MS/MS protein identification. This depletion method proved to be faster and more cost-effective than antibody-based methods, and could be helpful for biomarker enrichment and detection in medical research.