This study aimed to investigate a methodology for discriminating viable and non-viable T. gondii oocysts in water. Analyses included two steps: (i) microscopic investigation with vital dyes; (ii) molecular investigation, using a real time PCR (qPCR), after parasite treatment (or not) with propidium monoazide (PMA). The method was called qPCR-PMA. Oocyst aliquots were incubated (15 min) at 25 ºC or 100 ºC and analyzed by microscopy, after trypan blue and neutral red staining. Microscopic investigation determined viable and non-viable oocysts. For the molecular investigation, both aliquots of oocysts were treated with PMA. Non-viable oocysts, after PMA treatment, exhibited an inhibition of DNA amplification by qPCR. Although analyses were carried out with oocysts treated experimentally, these results suggest that qPCR-PMA can be a useful strategy to distinguish viable and non-viable T. gondii oocysts in water safety testing, showing if water is safe to drink.