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Infectious porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, but noninfectious PRRSV cannot. PCR and ELISA are commonly used for PRRSV detection but they cannot discriminate PRRSV infectivity. Virus isolation is a gold standard to determine virus infectivity. However, it is time‐consuming. Therefore, we developed a propidium monoazide (PMA) qPCR assay for rapid and universal detection of infectious PRRSV in this study. After comparing the inactivation efficacies of distinct disinfectants, ultraviolet (UV) light, and heat, heat at 72°C for 15 min was determined as an effective strategy for PRRSV inactivation, which was confirmed by virus isolation and immunofluorescence assay (IFA) detection. In addition, PMA pretreatment parameters were optimized, including PMA concentration (5 μM), PMA binding time (25 min), PMA binding temperature (37°C), and photolysis time (25 min). The optimal concentration of primers and probes adapted from our previous study was redetermined. The optimized PMA‐qPCR assay exhibited satisfied specificity, sensitivity, and reproducibility. Furthermore, the new PMA‐qPCR was applied on the detection of 270 clinical samples (including 57 environmental feces, 177 lungs, 33 lymph nodes [LN], and 3 sera) and compared with previously developed qPCR. Eighty samples were qPCR positive, while only 63 samples were PMA‐qPCR positive. No virus could be isolated in the 17 qPCR‐positive but PMA‐qPCR‐negative clinical samples; meanwhile, PRRSV could be isolated in representative PMA‐qPCR‐positive samples, supporting that only live PRRSV isolates in distinct samples could be detected by this PMA‐qPCR assay. In conclusion, this study provides the first PMA‐qPCR assay for rapid and universal detection of infectious PRRSV, offering an alternative and effective method for PRRSV diagnosis, prevention, and control. [ABSTRACT FROM AUTHOR]