Two‐dimensional liquid chromatography separation (2‐DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two‐dimensional gel electrophoresis (2‐DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0–8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix‐assisted laser desorption/ionization‐time of flight/mass spectrometry analysis for identification. The results showed that the 2‐DL provides high reproducibility of two‐dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2‐DE gels. In addition, this system also allows to separate particularly proteins with 40–9 kDa molecular weight.