Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible in an automated manner. The problem is difficult, as MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident, as phosphorylation sites are based on single peptide identifications, in contrast to protein identifications where at least 2 peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO2) selective phosphopeptide enrichment procedure for crude cell lysates, Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarised as follows: 1) enrichment for phosphopeptides by TiO2 chromatography, 2) dephosphorylation of half the sample 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation, to confirm phosphorylation, 5) comparison of MS/MS spectra before and after dephosphorylation, to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification is a clear improvement from identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large-scale analysis of phosphorylation sites in differentiating mouse myoblast cells.