Purpose: Despite recent progress in the proteomic analysis of peritoneal dialysate effluent (PDE), there remains unresolved problems in the development of an optimal sample preparation method. Experimental design: We examined five protocols for concentrating PDE proteins and the effects of immobilized pH gradient (IPG) strips with different pH ranges and sample loading techniques. In addition, we examined three kits for depleting high abundance proteins by SDS-PAGE and two-dimensional gel electrophoresis (2-DE). Results: PDE proteins precipitated with 75% acetonitrile (ACN) showed the greatest number of protein spots by 2-DE, with over 800 distinct spots. Higher-resolution images were obtained using IPG strips with a pH range of 4-7. The ProteoPrep immunoaffinity albumin and IgG depletion kit removed high abundance proteins with higher efficiency and more compatibility with isoelectric focusing (IEF). Removing high abundance proteins also increased the resolution and improved the intensity of low abundance proteins. Conclusion and clinical relevance: High-resolution 2-DE images of PDE proteins were obtained by concentrating samples with 75% ACN, using pH 4-7 IPG strips, and depleting high abundance proteins. This optimized method will enable future studies to discover predictive biomarkers of disease in patients on dialysis. (C) 2013 Elsevier Inc. All rights reserved.