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 Objetive: 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.