The ascomycete fungus Fusarium graminearum is a destructive fungal pathogen of wheat, barley and maize. Although reverse genetics and homologous recombination gene deletion methods have generated thousands of gene deletion mutants of F. graminearum, evaluating virulence of these fungal mutants is still a rate-limiting step. Here we present a protocol for inoculation of wheat coleoptiles with conidial suspensions for large-scale phenotypic analysis, and describe how it can also be used to assess fungal infectious growth and symptom developmentat a cellular scale. The inoculation method described in this protocol provides highly reproducible results in wheat coleoptile infection by F. graminearum.