The purpose of this study is to evaluate the diagnostic performance of the novel 2-photon excitation-based mariPOC© Assay (ArcDia Laboratories, Turku, Finland) for antigen detection of respiratory viruses versus real-time polymerase chain reaction (PCR). The mariPOC Assay and 2 multiplex real-time PCR techniques were performed on nasopharyngeal samples from pediatric patients with suspicion of acute respiratory infection admitted to a children's hospital in Spain during October 2011 to January 2013. A total of 233 samples were studied. Sensitivities and specificities (95% confidence interval) of the mariPOC Assay were for respiratory syncytial virus (RSV), 78.4% (69.7-85.6) and 99.2% (96.3-100.0); influenza virus (IFV) A, 66.7% (26.2-94.0) and 99.6% (97.9-100.0); IFV-B, 63.6% (33.6-87.2) and 100.0% (98.7-100.0); human metapneumovirus (hMPV), 60.0% (34.5-81.9) and 100.0% (98.6-100.0); adenovirus (ADV), 12.5% (0.6-48.0) and 100.0% (98.7-100.0), respectively. The mariPOC Assay is a highly specific method for simultaneous detection of 8 respiratory viruses but has sensitivities that range from moderately high for RSV to moderate for IFV and hMPV and low for ADV.