Multidrug-resistant tuberculosis (TB) threatens TB control worldwide. The microscopic observation drug susceptibility (MODS) assay is a low-cost, high-performance TB diagnostic tool for rapid liquid culture and direct isoniazid and rifampicin drug susceptibility testing (DST). The objective of this study was to explore the potential for extending the MODS assay to rapid second-line DST and to identify critical concentrations of candidate drugs for prospective testing. Sputum samples from 94 TB culture-positive patients receiving second-line TB agents were cultured following standardised MODS protocols, with a range of titrations of antimicrobial drugs added. Critical concentrations were determined using a modified Kaplan-Meier survival curve analysis. Candidate critical concentrations were determined for capreomycin (10 μg·mL-1), ciprofloxacin (1.25 μg·mL -1), cycloserine (40 μg·mL-1), ethambutol (10 μg·mL-1), ethionamide (5 μg·mL-1), kanamycin (5 μg·mL-1), para-aminosalicylic acid (10 μg·mL-1) and streptomycin (10 μg·mL -1). No cut-off point was identified for the other second-line drugs or for pyrazinamide. At particular concentrations of some second-line TB drugs this novel Kaplan-Meier analysis clearly differentiated populations that were susceptible or resistant. These candidate critical concentrations should now be tested in a range of epidemiological settings to define the performance of direct, second-line TB DST with MODS, offering potential low-cost second-line TB DST capacity. Copyright©ERS 2013.