【Abstract】 Objective To investigate the methods of urine sample collection and storage, concentrating of protein, fluorescence labeling and several key problems about two dimensional electrophoresis (2DE) for urinary proteomic research, and to provide the methodology for related clinical research. Methods Male patients with diabetes mellitus, hypertension, and less than 0.15g protein in 24 hour urine were included. Fresh urine samples were collected bedside. Urine samples were pooled together and were divided into 7 groups based on with and without adding cocktail protease inhibitors, storage temperature, protein desalting method, and types of immobilized pH gradient iso-electric focusing strips. 2-DE was used for comparison and analysis of samples from the groups. A part of the samples were analyzed by 2D fluorescence differential gel electrophoresis (2D-DIGE). Results From the 2-DE results, the number of protein spots was more in urine samples treated with protease inhibitors, stored at -80℃ and desalted by repeated ultrafiltration. The nonlinear IPG strips with pH 4-7 and 24cm long or pH 3-10 and 11cm long were suitable for urine 2DE. Urinary 2D-DIGE could provide repeatable results useful for clinical and laboratory research after labeling the samples with various Cydyes. Conclusion 2D-DIGE may give ideal results of urinary proteomics for clinical research, if appropriate methodology was adopted.