Nucleotide-binding leucine-rich repeat protein (NLR) can specifically recognize effector proteins secreted by pathogens, thereby initiating a strong immune response in host plants. Currently, utilizing this resistance gene to improve crop varieties is the most economically effective strategy. However, traditional identification methods are time-consuming and labor-intensive. RNA interference (RNAi) is a sequence-specific gene-silencing mechanism mediated by double-stranded RNA. This chapter provides a detailed protocol using tobacco rattle virus-mediated virus-induced gene-silencing (VIGS) screening platform in Nicotiana benthamiana for the fast identification of host NLR gene involved in viral effector-activated hypersensitive response (HR), with the turnip mosaic virus-coded nuclear inclusion protein a protease (NIa-Pro) as an example, which has been shown to activate HR in Nicotiana benthamiana.