Serum protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) appears to be an important diagnostic tool for a whole range of diseases. Sensitivities and specificities obtained with this new technology often seem superior to those obtained with current biomarkers. However, reproducibility and standardization are still problematic. The present review gives an overview of the diagnostic value of protein profiles obtained with SELDI in studies on prostate and ovarian cancer. To identify aspects important for protein profiling, we compare and discuss differences in pre- and post-analytical conditions presented in the literature supplemented with some of our own data. Further progress in protein profiling as a diagnostic tool requires a more comprehensive description of technical details in all future studies.