Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis (R) HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis (R) HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (> 30 mg/L albumin in urine) renal disease.