Background: Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated. Methods: To remove the most abundant proteins from a human urine sample, GenWay™ Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies. Results: Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343±20.0 μg, 5.8%; 186.3±13.3 μg, 7.2%; 292±20.6 μg, 8.8% [mean± standard deviation (SD), CV%], for GenWay™, Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468±21, 331±7, 368±22 and 304±7 (mean±SD) for GenWay™, Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p<0.001 compared to the GenWay procedure. Conclusions: The sequential procedure of urine samples using multi-protein immuno-affinity depletion represents a valid tool for simplifying 2-DE analysis of the urine proteome. Particularly, the GenWay™ kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome. Clin Chem Lab Med 2010;48:531–5.