Plant nucleotide-binding leucine-rich repeat receptors (NLRs) induce cell death by recognizing effectors secreted by pathogens into host cells. NLR activity can be evaluated by measuring the level of cell death. In Nicotiana benthamiana, this is typically done by co-expression of an NLR and its corresponding effector in the leaves via agroinfiltration. However, agroinfiltration is not suitable for protein expression in rice leaves. Here, we describe a method for preparing protoplasts from rice leaves and conducting a cell death assay using polyethylene glycol (PEG)-mediated transfection. This assay can also be applied to evaluate the activity of other cell death-inducing proteins.