As one of the most widespread posttranslational modifications, protein phosphorylation has been shown to be involved in virtually every basic cellular process. Advances over the past five years in the phosphoproteomics field have shed considerable light on the scope and complexity of phosphorylation-based signal transduction pathways in eukaryotes. However, the identification of the kinases responsible for specific phosphorylation events continues to be a slow step in advancing our understanding of phosphorylation networks. Kinases phosphorylate targets that fall within the context of specific consensus sequences. Thus, one approach to identifying novel kinase-substrate relationships is to determine the phosphorylation motif targeted by each kinase. Current methods, however, are not suitable for proteome-wide analysis. To this end, we have adapted the peptide array approach initially developed by Hutti et al., which makes use of a positional scanning peptide library. The main advantage of their approach over other current methods is that kinases can be assayed for their specificity regardless of whether an in vivo substrate is already known. We optimized this peptide array approach to 1536-well format for more high-throughput analyses and applied it to globally screen the Saccharomyces cerevisiae kinome for consensus phosphorylation motifs. To date, motifs have been identified for 59 of the 122 yeast kinases. Our results have demonstrated that kinases exhibit a wide range of selectivities and that related kinases have similar, but different, motifs. In addition, novel motifs were identified for five kinases: Atg1, Cdc15, Mps1, Rim15, and Tos3. Most importantly, we have shown, through validation experiments with Prk1, a kinase involved in endocytosis and the regulation of the actin cytoskeleton, that the comprehensive determination of consensus phosphorylation motifs in this manner enables the identification of novel substrates. The significance of this work notably extends beyond the identification of novel kinase-substrate relationships. This work has, for the first time, provided a comprehensive view of phosphorylation specificity for a single organism. With the high degree of conservation of kinases and signaling pathways, we expect that this work will become a general resource for future studies of phosphorylation-based signal transduction in all eukaryotes.