Abstract: A few protein post-translational modifications (PTMs), such as phosphorylation and ubiquitination, are known to be critical in regulation of protein kinase activities. However, the roles of other PTMs have not been extensively studied in kinases. Development of a comprehensive description of all types of PTMs and discovering novel in vivo PTMs in low abundance represent major analytical challenges. Toward this goal, we have developed a strategy for systematic and accurate identification of the full-spectrum of PTMs in yeast protein kinases. Our strategy involves isolation of GST-fused kinase proteins, mass spectrometry analysis, and unrestrictive PTM identification by PTMap algorithm. Among the 30 purified yeast kinases, we identified 27 different types of PTMs, and 53 PTM sites, among which are 13 novel mass shifts that have not been previously reported, likely representing novel PTMs. These results represent a significant expansion of our current understanding of PTMs in kinases and suggest highly complex regulation of kinase function.