Alternaria brassicicola is a necrotrophic fungus that causes black spot disease of most plants in the Brassicaceae, including cultivated Brassica species and weedy Arabidopsis species. Since the concept of transformation constructs of linear minimal elements was developed (Cho et al., 2006), we have produced over 200 strains of loss-of-function mutants with an aid of selectable marker genes. Pathogenicity assays are a time-consuming step in screening pathogenesis-associated genes among targeted gene mutants. Here we describe a method for pathogenesis assays of A. brassicicola. Using this method, we have discovered pathogenesis-associated genes and were able to further characterize the functions of selected gene (Cho et al., 2013; Cho et al., 2012; Srivastava et al., 2012).