<p>Female mosquitoes transmit pathogens to people during blood feeding, leading to ~700 million infections annually. Females navigate toward humans by integrating multiple sensory cues while flying. Here we describe a behavioral assay system using automated video analysis to measure mosquito landing and walking on target areas emitting human body temperature, elevated CO₂, and human skin odor, and quantify preference indices for such behaviors. This host seeker tracking (HOST) assay can be adapted to measure landing and movement on a target of interest under different combinations and intensities of sensory stimuli. It is distinct from other host-seeking assays in that it allows the impact of thermal infrared radiation and convection to be analyzed individually. Mated female mosquitoes are transferred to specialized cages, allowed to recover, and then assayed. During each HOST experiment, an assay cage is lowered into an arena where females are presented with host-associated cues, and their landing and walking behaviors on targets of interest are video-recorded. After completing the assays, the videos are analyzed using custom scripts (V-HOST), which automatically determine landing and movement on the target areas, and preference indices. The HOST protocol uses interchangeable cages rather than direct release of mosquitoes into the behavior arena, which increases assay throughput, and prevents release of mosquitoes. The HOST assay employs commonly available materials and equipment, making it a readily accessible technique. Using this approach, an experimenter can perform ~24 individual technical replicates in a 4 h period (ZT0–ZT4) and quickly perform the video analysis (10–15 min).</p>