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Activation of mitogen activated protein kinases (MAPKs) is involved in many abiotic and biotic stress responses including plant defense. MAPK acitvation is based on the dual phosphorylation of threonine (T) and tyrosin (Y) residues (T-x-Y motif) in the activation loop of the MAPK protein. By determination of the phosphorylation status of a specific MAPK one can detect if the MAPK has been activated or not. This protocol describes how to analyze the phosphorylation status of Arabidopsis MAPKs MPK3 and MPK6 by using leaf disks, western blotting and a specific antibody (Figure 1). It can also be used for the analysis of MAPKs in other plant systems although some alterations regarding protein extraction might be necessary.