West Nile Virus (WNV) is an arthropod-borne flavivirus whose zoonotic cycle includes both mosquitoes and birds as amplifiers and humans and horses as dead-end hosts. In recent years WNV has been spreading globally and is currently endemic in Africa, The Middle East, India, Australia, central and southern Europe, and the Americas. Integrated surveillance schemes and environmental data aim to detect viral circulation and reduce the risk of infection for the human population emphasizing the critical role for One Health principles in public health. Approximately 20% of WNV infected patients develop West Nile Fever while in less than 1%, infection results in West Nile Neurological Disease. Currently, the diagnosis of WNV infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia. The recent emergence of Zika virus epidemic in America and Asia has added another layer of complexity to WNV diagnosis due to significant cross-reactivity between several members of the Flaviviridae family such as Zika, dengue, Usutu, and West Nile viruses. Diagnosis is especially challenging in persons living in regions with flavivirus co-circulation as well as in travelers from WNV endemic countries traveling to Zika or dengue infected areas or vise-versa. Here, we review the recent studies implementing WNV surveillance of mosquitoes and birds within the One Health initiative. Furthermore, we discuss the utility of novel molecular methods, alongside traditional molecular and serological methods, in WNV diagnosis and epidemiological research.