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Indonesia is one of 22 countries with a high incidence of TB in the world, particularly related to TB-HIV and MDR-TB cases. Contamination of  normal flora from nasopharyngeal tract is the main problem  to isolate Mycobacterium tuberculosis (MTB) from sputum. It is needed a safe solution to decontaminate without killing  MTB bacilli. N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) is compared with  modified Petroff’s  which is widely used in laboratories, to get better outcome. Of the 110 sputum samples were collected from suspected cases of Pulmonary TB. Decontamination using 2% NALC-NaOH and Modifed Petroff  (4% NaOH) was performed before AFB smear and culture on LJ medium. Each group was assessed for contamination and culture positive rate. The positive culture was validated using chromatography test for detected antigen MPT-64 and PNB. Result of this study showed that NALC-NaOH and  modified Petroff’s  methods did not significantly affect positivity rate of  Acid-Fast Bacillus (AFB) smear, 71% and 66% respectively. Contamination on culture was significantly (p=0,034) higher in samples treated with NALC-NaOH (21%) compared to Modified Petroff methods(13%). Proportion of positive culture on samples treated with NALC-NaOH was lower than Modified Petroff, 65% and 70% respectively with p value=1. Conclusion of this study showed that sputum  decontamination using Modified Petroff methods is  still more effective than  NALC-NaOH  to increase  positivity rate of MTB culture.  However, the two methods were not significantly different to get positivity result on the microscopic examination  of AFB.