The aim of the study was to establish a live/dead qPCR with propidium monoazide (PMA) that can quantitatively differentiate between viable/non-viable microorganisms in dental hard tissues. Human premolars (n = 88) were prepared with nickel-titanium instruments and incubated with E. faecalis (21 d). Subsequently, the bacteria in half of the teeth were devitalized by heat inactivation (100 °C, 2 h). The following parameters were tested: PMA concentrations at 0 µmol (control), 50 µmol, 100 µmol, and 200 µmol; PMA incubation times of 30 min and 60 min, and blue light treatment for 30 min and 60 min. The teeth were ground using a cryomill and the bacterial DNA was quantified using qPCR, ANOVA, and p = 0.05. The qPCR of the control group detected a similar number of avital 9.94 × 106 and vital 1.61 × 107 bacterial cells. The use of PMA inhibited the amplification of DNA from non-viable cells during qPCR. As a result, the best detection of avital bacteria was achieved with the following PMA parameters: (concentration, incubation time, blue light treatment) 200-30-30; 5.53 × 104 (avital) and 1.21 × 100.7 (vital). The live/dead qPCR method using PMA treatment is suitable for the differentiation and quantification of viable/non-viable microorganisms in dentin, as well as to evaluate the effectiveness of different preparation procedures and antimicrobial irrigants in other biological hard substances.