West Nile virus (WNV) is a mosquito-borne single-stranded RNA virus, which has relatively recently emerged as a blood transfusion and organ transplantation transmissible pathogen. Low levels of WNV (viremia) are found in asymptomatic blood transfusion or cell/tissue donors with an infection, which poses a health threat to recipients. Since the introduction of nucleic acid testing (NAT) in 2003, many changes have occurred in the field of WNV detection and diagnosis. This review will focus on the recent progress in the in vitro assays for rapid and accurate detection of WNV in blood and tissues.