Pyrazinamide (PZA) has an excellent sterilizing activity against semi dormant tubercle bacilli. When used in combination with Rifampicin the treatment has been shortened from 1year to 6 months. PZA is a pro drug which requires an acidic environment for its conversion into active form of Pyrazinoic acid. The in vitro drug susceptibility for PZA is not performed routinely as the culture medium should be acidified which in turn affects the growth of Mycobacterium tuberculosis. Currently, the Mycobacterial Growth indicator tube is considered to be the Gold standard for PZA drug susceptibility testing. In the present study, the performance of the Modified Wayne's enzymatic test which detects the presence of the PZase enzyme required for the conversion of PZA into its active form was assessed by comparing it with the MGIT. 130 Mycobacterium tuberculosis culture isolates were tested by both the methods and their results were compared. The Wayne's Enzymatic assay showed an overall efficiency of 97.69%. The assay can be a good substitute for the MGIT in resource limited settings as the test is performed using reagents and chemicals readily available in a functional TB culture and Drug Susceptibility Laboratory. © 2010 RJPBCS.