Protein phosphorylation controls many cellular processes and activities. One of the major challenges in the proteomic study of phosphorylation is the enrichment of substoichiometric phosphorylated peptides from complex mixtures. Titanium dioxide (TiO2)-based chromatography is now widely applied to isolate phosphopeptides because of its efficiency and flexibility. In this study, a novel TiO2 coated matrix assisted laser desorption ionization plate is presented and tested for the purification of phosphopeptides from complex mixtures. The novel feature of this approach is the deposition of a nanostructured TiO2 film on stainless steel plates by pulsed laser deposition (PLD). By using tryptic digests of alpha-casein, beta-casein, and other nonphosphorylated proteins, the successful enrichment of phosphopeptides was possible with this novel device, called T-plate, even when working in the low fmol range, making the sample ready for mass spectrometric analysis in few minutes.