A reliable mass fragmentographic determination method for acetazolamide levels in body fluids was developed to favor the pharmacokinetic study on the patients receiving multiple doses. The use of 2-(N-propionamido)-1,3,4-thiadiazole-5-sulfonamide as the internal standard permitted the internal standard to be added to the sample before the extraction, and enabled the single ion measurement at m/e 249. Single ion measurement at m/e 249 detected 25 pg of acetazolamide. Multiple ion detection at m/e 249, 264 and 278 favored more accurate identification of each compound on the chromatogram. The assay results of the samples from volunteers were consistent with the ones reported elswhere.