Despite attractiveness of urine for biomarker discovery for systemic and renal diseases, the confounding effect of the high abundance plasma proteins in urine, and a lack of optimization of urine protein recovery methods are bottlenecks for urine proteomics. Three methods were performed and compared for percentage protein yield, yield consistency, ease and cost of analysis: (i) organic solvent precipitation, (ii) dialysis/lyophilization, and (iii) centrifugal filtration. Urine samples were subjected to an immunoaffinity column to deplete high abundance proteins. Difference gel electrophoresis was performed to assess use of depletion strategy for detection of low abundance proteins. Urine from healthy volunteers (n = 10) and kidney transplant recipients with proteinuria (n = 11) were used. Centrifugal filtration performed best for analysis ease and yield consistency. Highest percentage yield was obtained from dialysis/lyophilization but was laborious and residual salt interfered with subsequent gel electrophoresis. Organic solvent precipitation was inexpensive, but suffered from varying yield consistency. Increased spot intensity for some low abundance and previously undetected proteins were noted after depletion of high abundance proteins. In conclusion, we compare the pros and cons of different protein recovery methods and reveal an increase in the dynamic range of protein detection after depletional strategy that could be critical for biomarker discovery, particularly with reference to processing human study samples from clinical trials.