The discovery stage of proteome profiling typically involves the comparison of different states of a cell or tissue. One approach utilizes fractionation of the proteome into intact proteins, followed by mass spectrometry (MS) analysis. This paper will describe the combined benefits of an advanced two-dimensional, liquid chromatographic fractionation system, the ProteomeLab™ PF 2D, with the ultra-high performance of MALDI-TOF MS analysis for proteomics. The first dimension separation is chromatofocusing where proteins are separated by pI and collected in fractions based on pH intervals. Upwards of 20 pI fractions are then separated in a second dimension by reversed-phase chromatography. From each 2nd-dimension run, 80-90 fractions can be collected. To accommodate the large number of fractions generated by the first two dimensions for subsequent MS analysis, the Biomek® FX Laboratory Automation Workstation was used as the interface between the ProteomeLab PF 2D and MS by spotting the MALDI plate with 2nd dimension fractions. The Biomek FX Laboratory Automation Workstation prepared and spotted the fractions from the 2nd dimension runs along with the appropriate matrix onto a 384-well format MALDI target. The target plate is analyzed directly by a MALDI-TOF MS, which measures the mass-tocharge ratio of the intact proteins as the third dimension.