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Several types of olfactometers have been used to evaluate mosquito responses to agents that mimic natural volatiles that repel or attract. The Y-tube olfactometer has been widely used to study repellents and attractants, while the high-throughput screening system assay has only been used to study repellents. Whether the high-throughput screening system assay is suitable for evaluating attractants is unknown. We evaluated the responses to four lactic-acid-based mixtures and two non-lactic-acid-based chemical lure candidates using the high-throughput screening system (HITSS) for three mosquito species (laboratory strains and field populations of both <i>Aedes aegypti</i> (L.) and <i>Culex quinquefasciatus</i> Say.; laboratory strain of <i>Anopheles minimus</i> Theobald) under laboratory-controlled conditions. HITSS assay results showed that KU-lure #1 elicited the greatest percent attraction for pyrethroid-resistant and -susceptible <i>Ae. aegypti.</i> KU-lure #6 elicited the strongest attractive response for pyrethroid-susceptible and -resistant <i>Cx. quinquefasciatus</i> and pyrethroid-susceptible <i>An. minimus</i>. The response to the lures from each species was independent of the pyrethroid susceptibility status (<i>Ae. aegypti</i>, <i>p</i> = 0.825; <i>Cx. quinquefasciatus</i>, <i>p =</i> 0.056). However, a significant difference in attraction to KU-lure #6 was observed between diurnal and nocturnal mosquitoes (<i>Cx. quinquefasciatus</i> vs. <i>Ae. aegypti</i>, <i>p</i> = 0.014; <i>An. minimus</i> vs. <i>Ae. aegypti</i>, <i>p</i> = 0.001). The laboratory-level HITSS assay effectively selects potential lure candidates. Because the host-seeking behavior differs between mosquito species, further studies are needed to develop species-specific attractants. Additional studies in semi-field screen houses using commercial traps are necessary to evaluate the accuracy of these laboratory assay results.