This study evaluates the viability of Giardia intestinalis cysts, Cryptosporidium spp. and Toxoplasma gondii oocysts, which may be found in water and food matrices subjected to contaminated water. Viability is a key factor to be considered in order to assess parasite infectivity. People living in developing countries are particularly at risk of contamination by these pathogens, which can cause severe diseases particularly in immunocompromised people. In this report, we describe methods combining the mechanical rupture of (oo)cysts and mRNA extraction in order to determine their viability by qRT-PCR. The targeted genes are beta-giardin, hsp70, and SporoSAG for the detection of G. intestinalis, Cryptosporidium spp. and T. gondii respectively. The proposed method is compared to in vivo infectivity tests specific of each parasite. Then, this qRT-PCR method is applied directly on parasite suspensions and subsequently on basil experimentally contaminated with the parasites. For each protozoan, the detection limit of the qRT-PCR method is 2 parasites per reaction (2 μL mRNA analyzed) on parasite suspensions, and 3 parasites per gram of basil matrix. Compared to in vivo methods, qRT-PCR can detect viable but not infectious parasites. This qRT-PCR method could represent an attractive alternative as a specific and sensitive tool for: i) rapid assessment of the risk of human contamination by G. intestinalis, Cryptosporidium spp. and T. gondii parasites; ii) evaluation of the efficiency of industrial process.