Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens, due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 mug/mL) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two timepoints: R1 (mean 10 days) and R2 (mean 13 days) for each PZA concentration. A consensus reference test (CRT), composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. One hundred and eighty-two samples were evaluated. The sensitivity and specificity for 400 mug/mL ranged from 76.9% - 89.7% and 93.0% - 97.9%, respectively, and for 800 mug/mL ranged from 71.8% - 82.1% and 95.8% - 98.6%, respectively. Compared to MGIT- PZA, our test showed a similar turnaround time (median 10 and 12 days for PZA sensitive and resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line anti-tuberculosis drugs. Further optimization of test conditions would be useful, in order to increase its performance.