Standard tuberculosis (TB) treatment includes an initial regimen containing drugs that are both rapidly bactericidal (isoniazid) and sterilizing (rifampin and pyrazinamide), and ethambutol to help prevent the emergence of drug resistance. Antagonism between isoniazid and pyrazinamide has been demonstrated in a TB treatment mouse model. Because isoniazid's bactericidal activity is greatest during the initial two treatment days, we hypothesized that removing isoniazid after the second day would increase the effectiveness of the standard regimen. To test this hypothesis, we developed a mouse model to measure the early bactericidal activity (EBA) of drug regimens designed to analyze the essentiality of both isoniazid and pyrazinamide during the first 14 d of therapy. Our results clearly indicate that discontinuation of isoniazid after the second day of treatment increases the EBA of standard therapy in the mouse model, whereas omitting pyrazinamide during the first 14 d was detrimental. Substitution of moxifloxacin for isoniazid on day 3 did not increase the EBA compared with only removing isoniazid after day 2. Our data show that a mouse model can be used to analyze the EBA of TB drugs, and our findings support pursuing clinical trials to evaluate the possible benefit of removing isoniazid after the first 2 treatment days.