Proteome/peptidome profiling of human urine is a promising tool for the discovery of novel disease-associated biomarkers. However, a wide range of preanalytic variables influence the results of proteome/peptidome analysis regardless of the method used. We present a validated pretreatment protocol, which allows standardization of preanalytic modalities and facilitates reproducible peptidome profiling of human urine by means of magnetic bead (MB) separation in combination with MALDI-TOF MS. Such a procedure is necessary for generating consistent and reliable data from which meaningful results may be obtained for biomarker discovery and general proteomic experiments.