Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a highly lethal infectious disease with a global impact, despite being both preventable and treatable [1]. One of the major challenges in tackling TB is the delayed diagnosis and the emergence of drug-resistant strains [2,3]. Traditionally, TB diagnosis has relied on conventional methods, including sputum smear microscopy and culture-based techniques, despite advances in molecular tests [4]. While culture methods have been the reference for TB detection, they are resource-intensive and slow, especially for drug susceptibility testing (DST) [5].

The rise of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizes the need for faster and more accessible diagnostic approaches [6], particularly phenotypic methods for determining drug susceptibility profiles and mutations not targeted by molecular methods [7,8]. Phenotypic testing is crucial for treatment monitoring as it distinguishes viable bacilli [9], unlike genotypic methods [10,11], ensuring an accurate assessment of treatment efficacy. Consequently, investing in the development and optimization of new phenotypic methods is crucial to address the evolving challenges of TB management and ensure timely treatment.

The use of ATP quantification has been a cornerstone in microbial viability assessment for decades, particularly for the detection of bacterial contamination, offering a rapid and sensitive means of measurement [12], [13], [14]. The ATP bioluminescence assay offers a lower limit of detection compared to fluorescence assays and, because it is directly influenced by the living cells, it provides an effective tool to explore the effects of stressful conditions and exposure to harmful substances [15], including antimicrobials. However, its application to MTB remains relatively unexplored.

In this context, our study aims to enhance the utility of ATP bioluminescence as a phenotypic method for MTB detection and drug susceptibility testing. Our primary objective was to optimize the ATP bioluminescence protocol using BacTiter-Glo™ Microbial Cell Viability Assay (Promega, WI, USA), known for its luciferin-luciferase formulation claimed to selectively extract ATP, generating a luminescent signal proportional to cell count [16]. Although widely assessed with various microorganisms, its validation with MTB is lacking. Thus, we provide a standardized protocol for future MTB investigations. Briefly, we aimed to investigate the impact of varying volumes of MTB suspension and reagent on assay sensitivity, evaluate ATP extraction methods, establish calibration curves linking RLU values with MTB concentrations, and elucidate strain-specific responses to antimicrobial agents, providing valuable insights into susceptibility and resistance patterns in MTB.