Despite the importance of phosphorylation-dependent signaling to cellular physiology, a comprehensive understanding of the phosphorylation networks and pathways governing cell signaling in higher eukaryotes is still lacking. Here, we developed a combined bioinformatics and protein microarray-based strategy to construct a high-resolution map of the human phosphorylation networks. To accomplish this task, we performed phosphorylation reactions with 289 human kinases on protein microarrays composed of 4,191 unique, full-length human proteins.