New separation-free assay methods for rapid antigen detection of influenza A and B viruses are presented. The methods employ dry-chemistry reagents and the recently developed TPX (two-photon excitation) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody on polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation-free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay specific reagents are dispensed into the assay wells already in the manufacturing process of the test kits. Performance of the assay methods was tested with nasopharyngeal specimens using TR-FIA (time-resolved fluoroimmunoassay) as a reference method. The results suggest that the new technique enables rapid detection of influenza virus antigens with comparable sensitivity and specificity to the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of three orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.