Rapid and accurate pathogen detection plays an important role in the containment of local disease outbreaks. We present lateral flow devices (LFDs) for rapid detection of West Nile Virus (WNV) nucleic acid. WNV is becoming a public health concern in Europe due to climate change (1).The LFDs contain DNA probes on the test line, which directly bind the target nucleic acid. DNA probes present on gold nanoparticle (AuNP)-DNA-horseradish peroxidase (HRP) conjugates bind a different region of the same nucleic acid, leading to an accumulation of AuNPs on the test line when WNV ssDNA is present. Excess AuNP-DNA-HRP conjugate is bound by DNA probes immobilized on the control line, confirming the validity of the rapid test. Application of 3,3',5,5'-Tetramethylbenzidin, an HRP substrate, resulting in a blue product, enhances signal intensity. With this setup, WNV ssDNA concentrations down to 10 pM (6.022*106 copies/µl) could be detected. In order to improve sensitivity, WNV ssDNA was isothermally pre-amplified with Recombinase Polymerase Amplification (RPA) within 25 minutes before application to the LFDs, which led to an LOD of 500 copies/reaction. In conclusion, we have developed a diagnostic procedure consisting of RPA preamplification combined with LFD detection for as little as 500 copies/reaction (WNV ssDNA). (1) Farooq, Z.; Sjödin, H.; Semenza, J. C.; Tozan, Y.; Sewe, M. O.; Wallin, J.; Rocklöv, J. European projections of West Nile virus transmission under climate change scenarios. One health (Amsterdam, Netherlands) 2023, 16, 100509. DOI: 10.1016/j.onehlt.2023.100509.