The increase in emerging and reemerging infectious diseases has underscored the need for the prompt monitoring of intact infectious viruses and the quick assessment of their infectivity. However, molecular techniques cannot distinguish between intact infectious and noninfectious viruses. Here, two distinct methodologies have been developed for the expeditious and dependable quantification of intact infectious H1N1 virus, and several experiments have been conducted to substantiate their efficacy. One method is ICA-qPCR, which combines cell absorption and a qPCR assay. The other method is PMA-qPCR, which uses propidium monoazide and a qPCR assay. The quantification limit is 100 CCID50/mL in ICA-qPCR following a 1.5-hour cell absorption or 126 CCID50/mL after a 15-minute incubation. For PMA-qPCR, the limit was 2512 CCID50/mL. The number of genome copies quantified by the ICA-qPCR and PMA-qPCR methods was strongly correlated with the infectious titer determined by the CCID50 assay, thereby enabling the estimation of virus infectivity. The ICA-qPCR and PMA-qPCR methods are both suitable for the identification and quantification of intact infectious H1N1 virus in inactivated samples, wastewater, and biological materials. In conclusion, the ICA-qPCR and PMA-qPCR methods have distinct advantages and disadvantages, and can be used to rapidly quantify intact infectious viruses. These methodologies can facilitate the identification of the presence of intact infectious viruses in wastewater or on pathogen-related physical surfaces in high-level biosafety laboratories or medical facilities. Furthermore, this methodology can be employed to detect other pathogens, particularly those with high pathogenicity