Identification of protein kinase targets and specific inhibition of individual kinase isoforms on the protein level in planta are important techniques to elucidate signal transduction pathways. The use of ATP-binding pocket mutants, the so-called gatekeeper mutants, that accommodate N(6)-enlarged nucleotides and kinase inhibitors has allowed a dramatic increase in kinase isoform selectivity. In this chapter, we describe protocols for the identification and mutation of the gatekeeper residue, radiolabeling of N(6)-modified nucleotides, analysis of protein targets by using [(32)P]-labeled N(6)-modified nucleotides, and in vivo inhibition of kinase activity combined with subsequent molecular readouts. The chapter includes alternative approaches for the described techniques, considerations for other kinases and recommendations toward a setup of a substrate labeling experiment using total cell lysate.