Objectives: This study aimed to establish a multiplex quantitative polymerase chain reaction (MQ-PCR) assay for 12 bacterial pathogens found in lower respiratory tract infection (LRTI) and to evaluate its performance in a cohort of 211 patients with LRTI. Methods: The study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the pilot study, we established the MQ-PCR and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency. In the clinical validation study, we obtained 211 sputum and/or bronchoalveolar lavage fluid (BALF) samples and detected pathogens by MQ-PCR. The MQ-PCR time was 3 h from sample collection to complete pathogen detection. Results: The limit of detection was 10 0 0 copies/ml, and the maximum efficiency was > 95%. When cutoffs of > 10 5 copies/ml in sputum and > 10 4 copies/ml in BALF were applied, the sensitivity, specificity, and positive and negative predictive values of the MQ-PCR were 77% (95% confidence interval ICI] 67-88%), 94% (95% CI 93-95%), 25% (95% CI 19-31%), and 99% (95% CI 99-10 0%), respectively. Conclusions: This study demonstrates that the new MQ-PCR assay is time-saving, more effective and sensitive, and brings us closer to mainstream adoption of quantitative molecular detection of bacteria. (c) 2022 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )