The effects of competitive modulators of insect nicotinic acetylcholine receptors (nAChRs), such as those of neonicotinoids on bee pollinators, have been—and still are—a subject of extensive research and debate. Recent studies on honey bee cytochrome P450 monooxygenases (P450s) identified CYP9Q3 as the key determinant driving insecticide selectivity for various chemical classes, including neonicotinoids. In this study, artificial diet overlay bioassays were conducted with transgenic Drosophila melanogaster (Diptera: Drosophilidae) strains expressing honey bee P450s, particularly CYP9Q2 and CYP9Q3. Flies were exposed to varying concentrations of eight different commercial nAChR competitive modulators, and mortality was assessed after 48 hours. Our results demonstrate a varying level of mortality depending on the transgene when compared to wildtype flies and underpins the importance of CYP9Q enzymes as molecular determinants of insecticide selectivity. We identified nicotine as the least effective compound when tested against wildtype flies. Nitenpyram was most effective against wildtype flies, while showing significantly lower toxicity to flies ectopically expressing honey bee CYP9Q3. In general, our findings underscore the utility of transgenic D. melanogaster as a model to study various aspects of insect toxicology and resistance. Future studies will expand on these results by exploring biochemical and molecular tools to further understand and evaluate the mechanisms of insecticide selectivity and the potential risks to bee pollinator health.