SETTING: A Mycobacteriology Reference Laboratory in Johannesburg, South Africa. OBJECTIVE: To determine the ability of FASTPlaqueTB-RIF, a rapid bacteriophage-based test, to correctly identify rifampicin susceptibility in clinical strains of Mycobacterium tuberculosis after growth in the Bactec 460 semi-automated liquid culture system. DESIGN: A comparative study of FASTPlaqueTB-RIF and conventional drug susceptibility methods, with selection bias to include sufficient rifampicin-resistant strains. RESULTS: Rifampicin susceptibility results were available for 133 strains of M. tuberculosis. Using the Bactec 460 method, 42 of these strains were rifampicin-resistant and 91 strains were rifampicin-susceptible. A further one strain was found to have a mutation in the rpoB gene which was strongly indicative of rifampicin resistance. Sensitivity, specificity and overall accuracy for the FASTPlaqueTB-RIF were respectively 100%, 98.8% and 99.2% for detection of rifampicin resistance; 95.3% (41/43) of the rifampicin-resistant strains were also resistant to isoniazid (multidrug-resistant). CONCLUSION: FASTPlaqueTB-RIF offers performance comparable to the Bactec 460 method, with results available within 2 days and without the need for specialised equipment. This makes FASTPlaqueTB-RIF a rapid test for rifampicin resistance suitable for widespread application. A combination of the FAST-PlaqueTB-RIF test with semi-automated liquid culture reduces the time required to report susceptibility results, enabling rapid and appropriate management of patients with MDR-TB. Rifampicin resistance was a good predictor of multidrug resistance in this population.