A novel assay procedure has been developed for the determination of sulfonamide drugs with the general formula ArSO2NH2 where Ar = aryl (either homocyclic or heterocyclic). The assay depends on the displacement of a spin-labeled sulfonamide from the active site of bovine carbonic anhydrase B and can detect concentrations of sulfonamide as low as 0.3 µg/ml. This technique can also be used to assay low concentrations of carbonic anhydrase in hemolysates of red blood cells without prior removal of the hemoglobin or isolation of the enzyme.