Summary Anti-cancer drug development involves enormous expenditure and risk. Key to the rapid and economic identification of novel, bioavailable anti-tumor chemicals is the use of appropriate in vivo tumor models suitable for large-scale screening. Using a Drosophila Ras-driven tumor model, we demonstrate that tumor overgrowth can be curtailed by feeding larvae chemicals with the in vivo pharmacokinetics essential for drug development and known efficacy against human tumor cells. We then develop an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the tumor model. In a proof-of-principle pilot screen of 2000 compounds we identify the glutamine analog, Acivicin, a chemical with known activity against human tumor cells, as a potent and specific inhibitor of Drosophila tumor formation. RNAi-mediated knockdown of candidate Acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible critical target of Acivicin-mediated inhibition. Thus, the pilot screen has revealed that Drosophila tumors are glutamine-dependent, which is an emerging feature of many human cancers, and has validated the platform as a powerful and economic tool for in vivo chemical screening. The platform can also be adapted for use with other disease models, thus offering wide spread applications in drug development.