A new strategy is presented for the separation of complex peptide mixtures using 2D planar electrochromatography (PEC) and thin-layer chromatography (TLC). Phosphorylated peptides migrate more slowly in the first dimension based upon their anionic phosphate residues, and certain predominantly acidic phosphopeptides even migrate in the opposite direction, relative to the bulk of the peptides. Phosphopeptides are further distinguished based upon hydrophilicity in the second dimension. This separation mechanism, which then permits a restricted region of the plate to be interrogated for the presence of phosphopeptides by MS. Analysis of phosphopeptides directly from the plates using matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) and tandem MS enabled peptide sequencing and identification. The nature of the original protein can then be discerned once all the peptide fragments have been fully characterized and identified. These somewhat newer analytical approaches represent a new and novel route for investigating peptidomic and proteomic, as well as other "omic" type samples.