ObjectiveMultidrug-resistant tuberculosis (MDR-TB) case finding progressively increased in Ethiopia mainly as a result of the utilization of World Health Organization (WHO)-endorsed rapid technologies including MTBDRplus assay. However, there is inadequate data on routine testing performance of the MTBDRplus assay. Consequently, the aim of the study was to assess the routine performance of the MTBDRplus assay in detecting MDR-TB at St. Peter’s TB Specialized Hospital.ResultsThe sensitivity and specificity of MTBDRplus in detecting isoniazid (INH) resistance were 96.3 and 100%, respectively. While for rifampicin (RIF) 100% was recorded for both. Similarly, a sensitivity of 97.96% and a specificity of 100% was measured for detecting MDR-TB. Among 49 MTBDRplus RIF resistant isolates, 46 (93.9%) strains had rpoB mutation. S531L was the most common rpoB mutant (81.6% of RIF resistant cases). All MTBDRplus INH resistant isolates (n = 52) had S315T1 katG mutation.