Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs, or MPKs), and ten genes encoding MAPK kinases (MAPKKs, or MKKs), the upstream kinases that activate MAPKs in the signaling cascades. They play critical roles in many different biological processes ranging from growth/development to response to environmental stimuli and pathogen invasion. T-DNA knockout lines are not currently available for all these genes. There is also functional redundancy at both MAPK and MAPKK levels. In addition, embryo lethality is associated with some double mutant combinations, which makes it difficult to investigate their specific functions in plants. In such situation, the use of RNA interference technology by which mRNA of interested gene is targeted by double-stranded RNA (dsRNA) for degradation and gene silencing provides a powerful tool for loss-of-function analyses. In this chapter, we describe the hairpin-RNA interference (hpRNAi) method we employed to silence MPK3/MPK6 and their upstream MKK4/MKK5 in the model plant Arabidopsis, with particular emphasis on the generation of hpRNAi constructs for single gene RNAi, tandem RNAi of two MAPKK genes, and tissue-specific RNAi.