Accurate drug susceptibility testing (DST) for Mycobacterium tuberculosis is highly important for both therapy guidance and surveillance of drug resistance. Although liquid medium DST methods are used increasingly and seem most efficient and fast, the high costs hamper widespread implementation. In addition, an inability to check the colony morphology of the growing bacteria is a disadvantage of these methods. Moreover, these methods discriminate only between susceptibility and resistance and do not determine the MIC. In this paper, we describe a low-cost, reproducible, high-throughput, proportional absolute concentration DST method. The method uses a concentration series of antituberculosis drugs, including pyrazinamide in 7H10 medium, distributed semiautomatically in 25-well plates. The performance of this 25-well DST method was evaluated by the World Health Organization and the International Union against Tuberculosis and Lung Disease in 10 rounds of proficiency testing regarding sensitivity, specificity, efficiency, reproducibility, and predictive value for resistance and susceptibility. The performance of the method for these characteristics was 100% for isoniazid and from 96 to 100% for rifampin, 91 to 100% for streptomycin, and 85 to 100% for ethambutol. The method was 100% reproducible for all four drugs. The levels of drug resistance and the MIC distributions for the first-line antituberculosis drugs were determined for all 7,956 M. tuberculosis strains isolated in The Netherlands from 1998 to 2005 and amounted to 7.5% for isoniazid, 1.4% for rifampin, 8.5% for streptomycin, and 1.0% for ethambutol. Pyrazinamide testing was successful for 7,026 (88.3%) of the isolates and showed a resistance level of 0.8%.