Nanomaterial-mediated plant genetic engineering holds promise for developing new crop cultivars but can be hindered by nanomaterial toxicity to protoplasts. We present a fast, high-throughput method for assessing protoplast viability using resazurin, a non-toxic dye converted to highly fluorescent resorufin during respiration. Protoplasts isolated from hypocotyl canola (Brassica napus L.) were evaluated at varying temperatures (4, 10, 20, 30 ˚C) and time intervals (1-24 h). Optimal conditions for detecting protoplast viability were identified as 20,000 cells incubated with 40 µM resazurin at room temperature for 3 h. The assay was applied to evaluate the cytotoxicity of silver nanospheres, silica nanospheres, cholesteryl-butyrate nanoemulsion, and lipid nanoparticles. The cholesteryl-butyrate nanoemulsion and lipid nanoparticles exhibited toxicity across all tested concentrations (5-500 ng/ml), except at 5 ng/ml. Silver nanospheres were toxic across all tested concentrations (5-500 ng/ml) and sizes (20-100 nm), except for the larger size (100 nm) at 5 ng/ml. Silica nanospheres showed no toxicity at 5 ng/ml across all tested sizes (12-230 nm). Our results highlight that nanoparticle size and concentration significantly impact protoplast toxicity. Overall, the results showed that the resazurin assay is a precise, rapid, and scalable tool for screening nanomaterial cytotoxicity, enabling more accurate evaluations before using nanomaterials in genetic engineering.