Conservation of host signaling pathways and tissue physiology between Drosophila melanogaster and mammals allows for the modeling of human host-pathogen interactions in Drosophila. Here we present the use of genetically tractable Drosophila models of bacterial pathogenesis to study infection with the human opportunistic pathogen Pseudomonas aeruginosa. We describe and compare two protocols commonly used to infect Drosophila with P. aeruginosa: needle-pricking and injector-pumping. Each model has relevance for examining host components and bacterial factors in host defense and virulence. Fly survival and bacterial proliferation within host flies can be assessed as a measure of host susceptibility and pathogen virulence potential. The profiles of host responses toward P. aeruginosa virulent and non-virulent strains can be determined, enabling the identification of interaction-specific genes that could potentially favor or limit the initiation and progression of infection. Both of the protocols presented herein may be adapted for the inoculation and study of other microbial pathogens. P. aeruginosa cell preparation requires 24 h, fly inoculation 1 h, and fly survival and bacterial proliferation 1-4 d.