Sentinel chickens are used to detect western equine encephalomyelitis, St. Louis encephalitis, and West Nile virus activity. Flocks that receive high mosquito exposure will be most effective for surveillance purposes. However, mosquito population indices at the flock sites may only provide an indirect measure of potential exposure. Therefore, we developed an indirect enzyme immunoassay to detect chicken antibodies to salivary gland antigens (SGAs) from Culex tarsalis, the primary arbovirus vector in California. Chickens fed upon by Cx. tarsalis developed an antibody response that was proportional to the amount of exposure. Cross-reactivity between sera from Cx. tarsalis–exposed chickens and SGAs from Culex pipiens quinquefasciatus, Culex pipiens pipiens, Ochlerotatus melanimon, and Ochlerotatus sierrensis was likely due to shared SGAs among these species. This serologic assay for mosquito exposure could be used to evaluate the sensitivity of sentinel flocks for detecting arboviral activity.