doi:10.1016/j.xpro.2025.103643engRabodoarivelo, Marie SylvianneHoffmann, EikGaudin, CyrilAguilar-Ayala, Diana AngélicaGalizia, JordanaSonnenkalb, LindsayDal Molin, MichaelCioetto-Mazzabò, LauraDegiacomi, GiuliaRecchia, DeborahRybniker, JanManganelli, RiccardoPasca, Maria RosaliaRamón-García, SantiagoLucía, AinhoaProtocol to quantify bacterial burden in time-kill assays using colony-forming units and most probable number readouts for Mycobacterium tuberculosisART-2025-143165Here, we present a protocol to perform a time-kill assay (TKA) to quantify bacterial burden at multiple time points using colony-forming units and most probable number readouts simultaneously. We describe steps for preparing inoculum, experimental conditions, and sampling bacterial counts. We then detail procedures for quantification and analysis. TKAs provide longitudinal data reflecting the dynamics of the antibiotic effect over time against a planktonic culture and quantify the concentration-effect relationship.Here, we present a protocol to perform a time-kill assay (TKA) to quantify bacterial burden at multiple time points using colony-forming units and most probable number readouts simultaneously. We describe steps for preparing inoculum, experimental conditions, and sampling bacterial counts. We then detail procedures for quantification and analysis. TKAs provide longitudinal data reflecting the dynamics of the antibiotic effect over time against a planktonic culture and quantify the concentration-effect relationship.2025http://zaguan.unizar.es/record/15153310.1016/j.xpro.2025.103643http://zaguan.unizar.es/record/151533oai:zaguan.unizar.es:151533info:eu-repo/grantAgreement/EC/H2020/853989/EU/EUROPEAN REGIMEN ACCELERATOR FOR TUBERCULOSIS/ERA4TBThis project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 853989-ERA4TBSTAR Protocols 6, 1 (2025), 103643 [18 pp.]by-nc-ndhttps://creativecommons.org/licenses/by-nc-nd/4.0/deed.esinfo:eu-repo/semantics/openAccess