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Summary: Nucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNCP. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells. The effect of different types of antibiotics on the bacterial viability or viable but nonculturable (VBNC) state have been tested using both PMA‐qPCR and pre‐rRNA analysis with Pseudomonas aeruginosa. Our results showed that these two molecular methods could successfully eliminate false positive signals derived from antibiotics‐inactivated cells. [ABSTRACT FROM AUTHOR]