This study investigates the intrinsic substrate specificity of the human tyrosine kinase (Tyr kinase) family, revealing that these enzymes have distinct preferences for amino acid sequences around phosphorylated tyrosine (Tyr) residues. Using combinatorial peptide arrays, researchers profiled the sequence specificity of all 78 conventional Tyr kinases, identifying 15 distinct clusters based on their amino acid motif selectivity. These clusters span a continuum from acidophilic kinases that prefer negatively charged residues to basophilic kinases that favor positively charged residues. The findings suggest that Tyr kinase specificity has remained largely unchanged from worms to humans, indicating evolutionary conservation of their substrate recognition. The study also demonstrates that the intrinsic substrate specificity of Tyr kinases can be used to identify which kinases are most likely to phosphorylate specific Tyr sites. This information was validated using mass spectrometry phosphoproteomic datasets, accurately identifying dysregulated kinases in cells under various conditions. Furthermore, the topology of known Tyr signaling networks naturally emerged from comparisons of kinase sequence specificities and SH2 phosphotyrosine-binding domains. The research highlights the importance of understanding Tyr kinase specificity for developing targeted therapies. The comprehensive collection of Tyr kinase motifs enables the assessment of any Tyr site for its suitability as a substrate of each Tyr kinase, facilitating predictions about which kinases might phosphorylate it. These predictions correctly identify known substrates and nominate new putative substrates, providing insights into kinase regulation and dysregulation in various cellular contexts. The study also reveals that over 30% of the human Tyr phosphoproteome consists of sites poorly matched by the optimal motif specificity of canonical Tyr kinases. These sites may require induced proximity mechanisms such as RTK dimerization or SH2-domain-pTyr interactions for phosphorylation. The findings underscore the complexity of Tyr kinase signaling and the need for further research into the rules governing phosphoproteomic networks. The results provide a valuable resource for understanding the functional organization of the human Tyr kinome and its role in cellular communication and disease.This study investigates the intrinsic substrate specificity of the human tyrosine kinase (Tyr kinase) family, revealing that these enzymes have distinct preferences for amino acid sequences around phosphorylated tyrosine (Tyr) residues. Using combinatorial peptide arrays, researchers profiled the sequence specificity of all 78 conventional Tyr kinases, identifying 15 distinct clusters based on their amino acid motif selectivity. These clusters span a continuum from acidophilic kinases that prefer negatively charged residues to basophilic kinases that favor positively charged residues. The findings suggest that Tyr kinase specificity has remained largely unchanged from worms to humans, indicating evolutionary conservation of their substrate recognition. The study also demonstrates that the intrinsic substrate specificity of Tyr kinases can be used to identify which kinases are most likely to phosphorylate specific Tyr sites. This information was validated using mass spectrometry phosphoproteomic datasets, accurately identifying dysregulated kinases in cells under various conditions. Furthermore, the topology of known Tyr signaling networks naturally emerged from comparisons of kinase sequence specificities and SH2 phosphotyrosine-binding domains. The research highlights the importance of understanding Tyr kinase specificity for developing targeted therapies. The comprehensive collection of Tyr kinase motifs enables the assessment of any Tyr site for its suitability as a substrate of each Tyr kinase, facilitating predictions about which kinases might phosphorylate it. These predictions correctly identify known substrates and nominate new putative substrates, providing insights into kinase regulation and dysregulation in various cellular contexts. The study also reveals that over 30% of the human Tyr phosphoproteome consists of sites poorly matched by the optimal motif specificity of canonical Tyr kinases. These sites may require induced proximity mechanisms such as RTK dimerization or SH2-domain-pTyr interactions for phosphorylation. The findings underscore the complexity of Tyr kinase signaling and the need for further research into the rules governing phosphoproteomic networks. The results provide a valuable resource for understanding the functional organization of the human Tyr kinome and its role in cellular communication and disease.