Proteomics demands multidimensional separations, which can provide higher resolutions for complex biosystems.Two dimensional liquid chromatography, 2D LC, is playing an important role in modem proteomics due to its high level of instrumentalisation and automation.Although the analytical throughput of 2D LC has been greatly improved compared with classical two dimensional gel electrophoresis, it is still common to take a whole working day or so to complete a 2D LC run.Herein, based on high speed separations developed on an ultra high pressure liquid chromatography system, UHPLC, we evaluated high speed 2D LC mode.With a high flow rate of 5 ml/min at elevated temperatures, a reversed phase LC gradient, which took 25 min to finish in normal LC mode, was fulfilled within 4 min.An efficient peak generation rate of 8.5 peaks/min was achieved in UHPLC mode, compared with 2.4 peaks/min in HPLC mode.With tryptic digests as the sample, 2D maps obtained with UHPLC-and HPLC-based 2D separation were compared and the results represent significance of UHPLC in enhancing analytical throughput in screening proteomics.