Thin-layer chromatography (TLC) of peptides has not been widely implemented, mainly because alternative techniques are readily available for peptide analysis, such as SDS-polyacrylamide gel electrophoresis, capillary electrophoresis and peptide mass fingerprinting by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). Pressurized planar electrochromatography (PPEC) offers much higher separation efficiency and more rapid analyte fractionation in comparison to conventional TLC. Peptide separations by PPEC result from a combination of electroosmotic flow of the mobile phase and electrophoresis of the zwitterionic analytes. A newly developed instrument is presented that enables 85 mm square planar chromatography plates to be uniformly pressurized to 20-30 psi and run by high-voltage PPEC. This plate format is directly compatible with MALDI-TOF MS and MS/MS, conforming to most existing target plate standards. Typically, conventional TLC of peptides has only provided information pertaining to the retardation factor (Rf) of a peptide, which is not sufficient for the unambiguous assignment of sequence information. However, MALDI-TOF MS and MS/MS analysis of peptides directly from the silica surface of the TLC plate after PPEC separation can be performed with high m/z accuracy, enabling peptide sequencing and identification, as well as localization of phosphorylation sites.