A comparative study on the microbiological and spectrophotometric methods for estimation of rifampicin in urine was carried out in 15 individuals. The urinary levels of rifampicin were determined on 2nd, 8th and 15th days at 3 hour, 6 hour and 24 hour samples by the above methods after administration of 600 mg rifampicin. The results suggest that the microbiological assay is more sensitive than spectrophotometric method. The difference was highly significant in all the cases by paired t-test. Incidentally it was also noticed that urinary excretion of rifampicin was comparatively more on 15th day.