Synthetic West Nile virus RNA can be used for assay development, verification, and validation as well as monitoring of day-to-day test variation and lot-to-lot performance of molecular-based assays. The quantitative format allows for the generation of a standard curve for quantitative PCR (qPCR) to determine viral load. This preparation includes fragments from the 5’ UTR, capsid, membrane glycoprotein precursor (prM), Envelope protein (ENV), Nonstructural protein NS1, Nonstructural protein NS2A, Nonstructural protein NS3, RNA-dependent RNA polymerase NS5, and 3’ UTR regions.