A simple and reliable method for quantifying Fusarium head blight (FHB), a widespread disease of barley, would enhance our capacity in identifying resistance sources and highly aggressive isolates. A detached head assay (DHA) was used to reliably assess: (i) resistance of two barley cultivars, Arabi Aswad (AS) and Arabi Abiad (AB) with different susceptibility to FHB and (ii) aggressiveness in a set of 16 fungal isolates of four Fusarium species. The two inoculated cultivars showed different responses in FHB incidence (DI) and severity (DS) using spray and point inoculation on detached barley heads, respectively. On AB, susceptible under several experimental conditions, inoculation with different Fusarium species resulted in significantly higher DI and DS, compared with AS, which showed Fusarium resistance. Furthermore, the values of DI and DS were significantly correlated with the previous findings generated under several experimental conditions. The use of this simple and reliable method in barley breeding programs can speed up the process of identification of sources of resistance to multiple FHB isolates. To our best knowledge, this is the first in-depth report investigating the usefulness of DHA for distinguishing susceptibility of barley plants and aggressiveness of diverse Fusarium species from a breeder's point of view.