Arabidopsis thaliana (At), as the plant model organism, has the advantage of a bank of genotypes in stock centres worldwide. This allows for the assessment of genotypes and their contributes to disease resistance and/or susceptibility to a wide range of pathogens. We found that, through utilising the SALK lines (Alonso et al., 2003) provided by NASC (Nottingham, UK), to ensure reproducible disease phenotypes for disease analysis required additional specific steps not detailed in the current literature. This is likely due to the specificity of Fusarium graminearum (Fg) infection to the floral tissues of small grain cereals and the fact that At is a non-host of Fg. Whilst this protocol has been written and tested for the assessment of detached At leaves infected with Fg, it may be adapted for use for exploring other plant-pathogen interactions. This protocol is designed for the assessment of disease on Arabidopsis thaliana leaves by the fungal pathogen Fusarium graminearum. The nuances of this protocol that have been troubleshot have encouraged us to publish this as its own entity. The protocol described has been adapted from previous protocols published by Machado-Wood et al. (2021) and Chen et al. (2003), as well as by taking several components from Cuzick et al. (2008). Loosely, this protocol details seed sterilisation and plant growth, inoculations and diseases assessment.