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Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different steps of a rapid, automatic and quantitative image analysis to evaluate leaf area infected by Fusarium graminearum.