Abstract This project addresses an important limitation of existing culture-based markers used in Phase 2b/c tuberculosis (TB) trials that determine which regimens advance to Phase 3 testing. Unfortunately, the existing legacy marker of treatment effect (sputum culture) predicts clinical outcomes poorly and fails to detect residual M. tuberculosis (Mtb) that leads to relapse. Consequently, there is a critical unmet need for improved pharmacodynamic (PD) markers to de-risk clinical trial transitions. The over-arching objective of this project is to advance a novel non-culture assay of treatment effect called the RS ratio®. Unlike culture which estimates Mtb burden, the RS ratio demonstrates Mtb activity by quantifying ongoing ribosomal RNA (rRNA) synthesis based on the abundance of short-lived Mtb precursor rRNA. Murine studies show that rapid and profound suppression of the RS ratio is associated with faster cure. Additionally, the RS ratio is extremely sensitive, enabling detection of Mtb activity beyond the limits of culture. Results in human sputum indicate that detection of measurable residual disease (MRD) (i.e., positive RS ratio but negative sputum culture) at the end of treatment is associated with subsequent microbiologic relapse. The RS ratio assay is being developed by an academic collaboration called the Consortium for Applied Microbial Metrics (CAMM). Five key objectives have been included to support the acceleration of the RS ratio towards practical application as a PD marker for ACTG TB trials. Objective 1 is achieving Clinical Laboratory Improvement Amendments (CLIA) certification for the CAMM laboratory to assure that the RS ratio meets regulatory standards. Objective 2 is completing analytical method validation of RS ratio assay that will formally assessing the performance characteristics and the optimal conditions for that will generate the reproducibility and accuracy of the assay. Objective 3 is external laboratory replication of RS ratio assay in conjunction with the NIH TBQA through a multi- step process of transferring technology and assessing between-lab concordance. Objective 4 is development of a statistical analysis plan and initiation of the FDA Biomarker Qualification Pathway which will rigorously define a precise context of use for the RS ratio in Phase 2 TB trials. Objective 5 will compare the RS ratio with conventional PD markers in a recently completed Phase 2a trial to further document the performance characteristics of assay relative to existing PD markers. Collectively, this body of work will advance a novel PD marker that can accelerate and de-risk development of more effective TB treatments.