Urine has garnered tremendous interest over the past decade as a potential source of protein biomarkers for various pathologies. However, due to its low protein concentration and the presence of interfering compounds, urine constitutes a challenging analyte in proteomics. In the context of a project aimed at the discovery and evaluation of new candidate biomarkers of bladder cancer in urine, our laboratory has implemented and evaluated an array of preparation techniques for urinary proteome analysis. We present here the protocol that, in our hands, yielded the best overall proteome coverage with the lowest analytical effort. It begins with protein precipitation using trichloroacetic acid, in solution digestion and RP-C18 cartridge desalting of the resulting peptides mixture, and is followed by peptide fractionation by gel-free isoelectric focusing, and nano-LC-MS/MS for database compilation.