Mammalian hosts have traditionally been considered the "gold standard" models for studying pathogenesis and antifungal drug activity in invasive aspergillosis (IA). Nevertheless, logistical, economical, and ethical constraints make these host systems difficult to use for high-throughput screening of putative Aspergillus virulence factors and novel antifungal compounds. Here, we present Drosophila melanogaster, a heterologous non-vertebrate host with conserved innate immunity and genetic tractability, as an alternative, easy-to-use, and inexpensive pathosystem for studying Aspergillus pathogenesis and antifungal activity. We describe three different infection protocols (i.e., injection, rolling, ingestion) that introduce Aspergillus conidia at different anatomical sites of Toll-deficient Drosophila flies. These reproducible assays can be used to (1) determine the virulence of various Aspergillus strains and to (2) assess the anti-Aspergillus activity of orally absorbed antifungal agents in vivo. These methods can also be adapted to study pathogenesis and antifungal drug activity against other medically important human fungal pathogens.