A new strategy is presented for the separation of complex peptide mixtures using a combination of electrically-driven planar chromatography (PEC) and thin-layer chromatography (TLC), followed by direct detection of analytes using MS. Visualization of phosphopeptides from the 2D separations, using a phosphorylation-sensing dye, facilitated system optimization. Fluorescence imaging was achieved using a xenon-arc lamp-based charge-coupled device (CCD) camera system. Using the new 2D system, phosphorylated peptides migrate in the opposite direction as the bulk of the other peptides in the first dimension, based upon charge properties and are further distinguished from adventitial peptides based upon hydrophilicity in the second dimension. This permits a restricted region of the plate to be interrogated for the presence of phosphopeptides by mass, as demonstrated with tryptic digests of ovalbumin, riboflavin-binding protein, heat shock protein 90, beta-casein and alpha-casein. Analysis of phosphopeptides directly from the plates using orthogonal MALDI-TOF MS was demonstrated. With this instrument, ionization and mass analysis are decoupled. Consequently, mass analysis is not affected by spatial variations and sample topography, allowing plates to serve as the sample target. The newly developed phosphopeptide mapping approach should allow routine identification of phosphopeptides, without the use of radioisotopes or surrogate dyes. The cited multidimensional approach offers certain performance advantages relative to currently practiced on-line methods, including ease of use, ability to fractionate larger amounts of sample, ability to re-analyze fractions and the ability to use off-the-shelf equipment that requires little or no instrumental modification.