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Comprehensive analysis of protein kinases is reviewed.Mass spectrometry-based phosphoproteomics have been applied to profile the protein kinases.Array-based approaches as well as other tools have been also used to determine the substrates of protein kinases.Protein phosphorylation mediated by protein kinases plays key roles in cellular signal transduction in both eukaryotic and prokaryotic organisms. Many researches have been conducted to investigate their functions and regulatory mechanisms, but these were mostly focused on particular kinases related to well-known signaling pathways, drug targets, diseases, and so on. Therefore our overall understanding of the whole set of kinases (the kinome), including their structural basis for substrate recognition, is still limited. In this review, we describe technologies for determination of sequence preference and physiological substrates of individual target kinases and for kinome-scale profiling, focusing on peptide array technology or proteomic approaches based on liquid chromatography-mass spectrometry (LC–MS).