The study profiles the substrate sequence specificity of all human tyrosine kinases using combinatorial peptide arrays, revealing a diverse range of optimal residue patterns around the phosphorylated tyrosine site. This analysis identifies kinases that can phosphorylate any tyrosine site and accurately predicts kinases dysregulated in various cellular conditions. The intrinsic substrate specificity of tyrosine kinases is conserved from worms to humans, suggesting evolutionary conservation. The findings enhance our understanding of tyrosine kinase signaling networks and provide a resource for predicting kinase activity in phosphoproteomic datasets.The study profiles the substrate sequence specificity of all human tyrosine kinases using combinatorial peptide arrays, revealing a diverse range of optimal residue patterns around the phosphorylated tyrosine site. This analysis identifies kinases that can phosphorylate any tyrosine site and accurately predicts kinases dysregulated in various cellular conditions. The intrinsic substrate specificity of tyrosine kinases is conserved from worms to humans, suggesting evolutionary conservation. The findings enhance our understanding of tyrosine kinase signaling networks and provide a resource for predicting kinase activity in phosphoproteomic datasets.