Mosquitoes that transmit human diseases are of major importance to the international public health community. Pesticides remain a major component of integrated programs to control these medically important species. However, very few types of pesticides are currently registered for mosquito control. A high-throughput screening method using first-instar larvae of Aedes aegypti was created and evaluated in our laboratory to quickly screen large numbers of chemicals for activity against mosquitoes. LC50 values of a representative group of compounds were determined using this high-throughput screening method and compared with LD50 values determined by topical application against female adults of Ae. aegypti. Our results show that this high-throughput screening method is suitable for screening large numbers of candidate chemicals quickly to identify effective compounds.