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A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a cell culture system coupled with four RTqPCR assays to detect four species of human enterovirus (e.g., Enterovirus A, Enterovirus B, Enterovirus C and Enterovirus D). Evaluation of the RTqPCR assays was conducted with coxsackievirus A10, echovirus 30, poliovirus 1 and enterovirus 70 and resulted in 100% specificity for the tested assays. A comparison of ICC-RTqPCR between the individual enteroviruses and a mixture of all four viruses resulted in an approximate 1:1 correlation, demonstrating a lack of competition during incubation in cell culture and RTqPCR. The simultaneous detection of multiple human enterovirus species within mixed cultures is relevant to many applications, including virus disinfection studies. This high-throughput, multiplex approach costs less in money and time. By helping with data collection, this approach will lead to more statistically sound data sets that directly compare the inactivation rates of enteroviruses tested.