This study aimed to establish a multiplex quantitative PCR (MQ-PCR) assay for 12 bacterial pathogens of lower respiratory tract infection (LRTI) and evaluate its performance by a cohort consisting of 211 patients with LRTI.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 through the limits of detection, repeatability, specificity, and efficiency. In the clinical validation study, we enrolled 211 sputa and/or bronchoalveolar lavage fluid (BALF) to detect pathogen by MQ-PCR. The MQ-PCR takes only 3 h from the sample obtained to complete pathogen detection.The limit of detection was 1000 copies/mL, and the efficiency could reach over 95%. When cutoffs of ≥105 copies/mL for sputum and ≥104 copies/mL for BALF were applied, the sensitivity, specificity, positive and negative predictive values of the MQ-PCR were 77% (95% confidence interval [CI], 67-88%), 94% (95% CI, 93-95%), 25% (95% CI, 19-31%), and 99% (95% CI, 99-100%), respectively.This study demonstrates that the new MQ-PCR assay is time-saving, more effective and sensitive, and brings us closer to the mainstream adoption of quantitative molecular detection of bacteria.Copyright © 2022. Published by Elsevier Ltd.