The recent outbreaks of West Nile virus (WNV) infection in the northeastern United States and other regions of the world have made it essential to develop efficient, sensitive, and rapid protocols for virus surveillance. Laboratory testing is the backbone of any surveillance program. Protocols to detect the presence of WNV have been refined since 1999 for sensitivity, speed, efficiency, and specificity. This paper presents the protocols currently used by the New York State Department of Health to handle vertebrate and mosquito specimens that have been submitted for WNV testing to the Arbovirus Laboratories of the Wadsworth Center.