The family of transmembrane receptor kinase is the largest protein kinase family in Arabidopsis. However many of these kinases have yet uncharacterized functions and little is known about direct substrates of these kinases. Here, we present a large-scale phosphoproteomics method involving label-free quantitation-based comparative phosphopeptide profiling of knockout mutants in receptor-like kinases. This approach, among other physiological and cell biological experiments, is one step in understanding the functional roles of plant kinases in the context of their signaling networks.