Resazurin microtitre assay (RMA) has been successfully used to detect minimal inhibitory concentrations (MICs) of both first-line and several second-line drugs in drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). In this study, we firstly compared prothionamide (PTH) susceptibility testing of Mycobacterium tuberculosis (MTB) using resazurin microtitre assay (RMA) and MGIT. Overall, the sensitivity and specificity of RMA for detecting PTH susceptibility was 96.5% [95% confidence interval (CI): 91.7–100.0] and 93.2% (95% CI: 89.6–96.8) respectively. In addition, the median time to positivity was significantly shorter for RMA than for the automated MGIT 960 (RMA, 8 days [range: 8–8 days] vs MGIT, 10.1 days, [range: 5.0–13.0]; P < 0.01). Concordance rate for MICs between RMA and MGIT for PTH-resistant group was 64.3% (95% CI: 46.5–82.0), which was significantly lower than that of PTH-susceptible group (85.9%, 95% CI: 78.8–93.0; P= 0.01). In conclusion, our data demonstrated that RMA can be used as an acceptable alternative for determination of PTH susceptibility with shorter turn-around time. When compared with MGIT 960, RMA method was prone to produce higher MICs for PTH-resistant MTB strains.