Disease biomarkers for diagnostic and prognostic purposes are most likely within an extremely low concentration range and are thus masked by the presence of high-abundance proteins. Therefore, removing high-abundance proteins is the main challenge for identifying disease biomarkers. In addition, the solution obtained from high-abundance protein depletion kits contains a rich array of compounds, which interfere with isoelectric focusing (IEF). In the present study, the effect of two commercial kits was evaluated and the downstream IEF protocol was optimized. High-resolution results could be obtained according to the following conditions: The ProteoPrep Blue Albumin and IgG Depletion kit depleted albumin and IgG; immobilized pH gradient strips (typically 18 cm) were rehydrated with sample buffer containing 250 mu g serum proteins at 30 v for 6 h, 60 v for 6 h, 200 v for 2 h, 500 v for 2 h, 1,000 v for 2 h, 5,000 v for 2 h, 10,000 v for 2 h and then focusing at 10,000 v up to 110 k vhs. In addition, the protein spots identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that all proteins had a low abundance. The present study not only provides a definite and effective method for removing high-abundance proteins, but also provides a proper protocol (protocol C) for downstream IEF. The present study includes a comprehensive investigation of serum proteomics, which paves the way for serum protein research.