Protein kinases are major regulatory components in almost all cellular processes in eukaryotic cells. By adding phosphate groups, protein kinases regulate the activity, localization, protein-protein interactions and other profiles of their target proteins. It is known that protein kinases are central components in plant response to environmental stresses such as drought, high salinity, cold and pathogen attack. Although the phosphorylation-dependent signaling plays critical roles in plant stress biology, only a few targets of these protein kinases have been identified. Moreover, how these protein kinases regulate the downstream biological processes and mediate the stress responses are still largely unknown. In this study, we introduce a novel strategy based on isotope-labeled in vitro phosphorylation reactions using in vivo phosphorylated peptides as substrate pools and apply this strategy to identify in putative substrates of nine protein kinases that function in plant abiotic and biotic stress responses. As a result, we identified more than 5,000 putative target sites of osmotic stress-activated SnRK2.4 and SnRK2.6, ABA activated protein kinases SnRK2.6 and Casein-Kinase Like 2 (CKL2), elicitor-activated protein kinase CDPK11 and MPK6, cold-activated protein kinase MPK6, H2O2-activated protein kinase OXI1 and MPK6, salt-induced protein kinase SOS1 and MPK6, as well as the low-potassium-activated protein kinase CIPK23. These results provide comprehensive information on the role of these protein kinases in the control of cellular activities and could be valuable resource for further study on the mechanism underlying plant response to environmental stresses.