MOTIVATION: A key challenge in phosphoproteomic studies is to distinguish functionally relevant phosphorylation sites from potentially 'silent' phosphorylation. Considering that relevant phosphorylation sites are expected to be better conserved during evolution than overall Serine, Threonine and Tyrosine (S/ T/ Y) residues, we asked whether this can be directly demonstrated through statistic analysis, using a large experimental dataset. RESULTS: Analyzing phosphoproteomic data derived from the human mitotic spindle apparatus, we found that 95.2% of 1744 phosphorylation sites are conserved in at least one of six other vertebrate species. Using a new score, termed conservation Z-score (CZ-score), we demonstrate that phosphorylation sites are significantly better conserved than other S/T/Y sites, a conclusion validated from several kinase consensus motifs. Most importantly, phosphorylation sites with experimentally verified biological functions were significantly better conserved than other phosphorylation sites, indicating that analysis utilizing evolutionary conservation may constitute a powerful basis for the development of improved phosphorylation site predictors. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.