Female mosquitoes respond to the world around them by using chemosensory organs, such as their antennae, to detect volatile compounds emitted from a vertebrate host. These chemosensory systems facilitate the interpretation of external stimuli from the periphery by connecting to the central nervous system and eliciting behaviors necessary for survival, such as obtaining a blood meal. This innate behavior leads to the transmission of pathogens, including dengue virus, chikungunya virus, and Zika virus. Olfaction is a primary sense mosquitoes use to differentiate between vertebrate hosts, and studying it can lead to novel strategies to reduce the risk of disease. In this protocol, we present an olfactory-driven behavioral assay using a uniport olfactometer that measures mosquito attraction rate to a specific stimulus. We include details of the behavioral assay and data analysis as well as how to prepare the mosquitoes before their introduction into the olfactometer. This uniport olfactometer behavioral assay is currently one of the most reliable methods to study mosquito attraction to a single stimulus.