Histone methylation is a central means by which gene expression is controlled. In the lower eukaryote, Saccharomyces cerevisiae, histone methylation is regulated by a reduced, but evolutionarily conserved set of methyltransferases (Set1, Set2, Set5, Dot1) and demethylases (Jhd1, Jhd2, Rph1, Gis1). While the catalytic activity and specificity of these enzymes have been established, knowledge of how they themselves are regulated by post-translational modification is surprisingly limited. Consequently, the regulatory network of histone methylation in yeast remains unknown and is also unknown in all other eukaryotes. To this end, we aimed to comprehensively characterise the modifications occurring on the eight yeast histone methyltransferases and demethylases in vivo. This was achieved by purification of these proteins, and their analysis by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). With respect to phosphorylation, to date, we have identified modification sites on the histone methyltransferases Set5 (nine sites) and Dot1 (three sites), and the demethylases Gis1 (seven sites) and Jhd2 (two sites). Fourteen of these sites validate those observed previously in high-throughput phosphorylation screens, while seven sites are novel. To determine the upstream kinases responsible for the phosphorylation, and potential regulation of these enzymes, mass spectrometric analysis was employed to monitor levels of histone methylation in kinase knockout yeast strains. As a proof of concept, quantification of H3K79 methylation in the knockout cells established twenty-five kinases that are not responsible for the regulation of Dot1 methyltransferase activity. The screening of all other non-essential kinases is in progress. We plan to extend this methodology to the other yeast histone methyltransferases and demethylases in order to comprehensively integrate these enzymes into intracellular signalling pathways, and ultimately facilitate the assembly of the first regulatory network of histone methylation in eukaryotes.