We standardized and assessed the performance of an in-house microtiter assay for determining the susceptibilities of Mycobacterium tuberculosis clinical isolates to isoniazid based on mycobacteriophage amplification technology. Seventy isolates (43 resistant and 27 sensitive according to the BACTEC 460 radiometric method and MIC determination) were studied. The isoniazid resistance molecular mechanism was previously determined by sequencing the entire katG gene and the mabA-inhA regulatory region. The sensitivity of the mycobacteriophage-based assay in detecting isoniazid resistance was 86.1%, the specificity achieved was 92.6%, and the overall accuracy was 88.6%. In order to assess the possible influence of resistance levels on the mycobacteriophage- based-assay sensitivity, the results were analyzed according to the isoniazid MICs. All the isolates exhibiting high-level resistance (MIC ≥ 2 μg/ml) were scored as resistant by the mycobacteriophage-based assay (100% concordance), and 95% showed mutations or deletions in the catalytic domain of the katG gene. In contrast, 26.1% of the low-level-resistance strains (MICs, 0.25 to 1 μg/ml) were misclassified, and 66.7% had alterations in the mabA-inhA regulatory region. The mycobacteriophage-based assay could be used as a rapid method to detect the isoniazid susceptibility pattern, although data from those areas with high rates of low-level-resistance strains should be interpreted with caution. The features of the assay make it suitable for widespread application due to its low technical demand and cost. Copyright © 2006, American Society for Microbiology. All Rights Reserved.