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Ascorbate (Vitamin C) fulfills various functions in plant photosynthesis and abiotic stress tolerance. The four key enzymes involved in the ascorbate-turnover pathway are ascorbate peroxidase, ascorbate oxidase, monodehydroascorbate reductase, and dehydroascorbate reductase. Several reports have shown the pivotal roles of these enzymes in plant development and stress tolerance. Therefore, reliable and rapid assay protocols are required for researchers to investigate their enzymatic activities during plant development and stress responses. Previously published methods for analyzing these enzymatic activities rely on cuvette spectrophotometers, which can only handle one sample per test, leading to a prolonged investigation. In this protocol, we employed a 96-well microplate reader to analyze at least eight samples with two technical replicates simultaneously. We analyzed two rice (Oryza sativa L.) genotypes with distinct ascorbate oxidase and dehydroascorbate reductase activities to demonstrate the assay process, including plant growth, sample preparation, reaction setup, and data analysis. Our protocol provides a high throughput method for investigating ascorbate turnover-related enzymatic activities in plants.