Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) has been recognized as an appropriate technology for biomarker discovery. Nevertheless, whenever human blood serum is studied, its sensitivity is restricted due to the wide dynamic range of serum protein concentrations. In this context, sample pre-fractionation targeting the low abundant proteins may help to overcome these limitations. Here, we evaluate the combination of ProteoMiner™ pre-fractionation and SELDI based protein profiling. We introduce a simplified workflow and demonstrate the high sensitivity and reproducibility of this combined profiling approach. Our analyses show that this combination is suitable for large-scale serum proteome profiling studies yielding reliable and reproducible results.