Background: Rapidly identifying candidate genes potentially involved in stress tolerance is crucial for functional genetic studies in non-model plant species. Agrobacterium-based transient assays can be conducted in many plants using fleshy tissues such as leaf/fruit/petal. However, such assays are not feasible in perennial ryegrass (Lolium perenne L.) and most other grass species since these plants have no fleshy tissue to be readily infected. Results: In this study, we developed a Protoplast-based Rapid stress regulatory gene Identification Assay (briefed as ‘PRIDA’) to rapidly identify genes in the regulation of plant heat and oxidative tolerance in perennial ryegrass. Using ryegrass mesophyll protoplasts, vectors harboring target genes or their RNAi hairpin structures driven under the maize ubiquitin promoter were transformed into protoplasts to transiently over-express or suppress the target genes. The transformed protoplasts were then subjected to optimized heat (35 to 37 ℃) or oxidative (50 to 75 mM H2O2) stress, and their viabilities were immediately recorded under a microscope after 0.1% Evans blue dye staining. The validity and reliability of PRIDA were verified by transiently knocking-down LpNOL/LpSGR and over-expressing LpTT3.1/LpTT3.2 that led to significantly compromised or enhanced protoplast viability after heat or H2O2-induced oxidative stresses. Finally, using ‘PRIDA’, we identified three heat-induced differentially expressed E3 ligase genes as potential regulators in plant heat and/or oxidative stress tolerance. Conclusion: This protoplast-based ‘PRIDA’ can be used as a versatile assay to rapidly identify and characterize candidate regulatory genes in heat and oxidative stress that will accelerate molecular genetic studies in perennial ryegrass. This assay can be adapted to other non-model plant species after adjusting abiotic stress parameters for further research and the development of improved cultivars.