We conducted a laboratory evaluation of the ability of commercial antigen-capture assays, the Rapid Analyte Measurement Platform (RAMP®) and the VecTest® wicking assay, as well as Real Time reverse transcriptase–polymerase chain reaction (RT-PCR, Taqman) and Vero cell plaque assay to detect West Nile virus (WNV) in large mosquito pools. Real-Time PCR (Taqman) was the most sensitive, detecting WNV ribonucleic acid (RNA) in 100% of samples containing a single infected mosquito in pool sizes of up to 500 mosquitoes. Mosquito body tissues minimally impacted the ability of Real Time RT-PCR to detect WNV in a pool size of 500, reducing sensitivity by 0.6 log₁₀ plaque-forming units (PFU)/ml. Vero cell plaque assay detected live virus from a single infected mosquito in 100% of pools containing up to 200 mosquitoes, but was unreliable at larger pool sizes. VecTest detected 100% of positive pools containing 50 mosquitoes with 5.8 log₁₀ PFU/ml virus, 100 mosquitoes with 5.9 log₁₀ PFU/ml, and 200 mosquitoes with 5.2 log₁₀ PFU/ml. The RAMP assay detected 100% of positive pools containing 50 mosquitoes with 3.3 log₁₀ PFU/ml virus, 100 mosquitoes with 3.7 log₁₀ PFU/ml, and 200 mosquitoes with 4.0 log₁₀ PFU/ml. Results indicate that WNV can be reliably detected by all 4 assays in pools of mosquitoes exceeding 50 specimens, though there is some loss of sensitivity with very large pool sizes.