Two approaches for sulfonamides (SA's) determination, based on carbonic anhydrase enzyme inhibition have been investigated in this work. In the first method, nine different SA's have been screened using simultaneous multiphotometric measurement in multiwell plates. The sulfanilamide (SAD) showed significant inhibition compared to other sulfonamides. The carbonic anhydrase (CA) kinetic interactions reveal noncompetitive binding of SAD. Interferences from other inhibitors with enzyme were studied and the results showed very good selectivity toward SAD. In the second approach, an electrochemical enzyme inhibition biosensor, based on CA entrapped in a carbon paste electrode using carbon black nanoparticles and solid paraffin, was successfully applied to SAD measurements. Results from the quantitative analysis of SAD are discussed in terms of detection limit, linear range and sensitivity using multiphotometric and biosensor-based methods The biosensor developed was successfully applied to the determination of SAD at submicromolar levels and it is recommended for application for in situ analysis. (C) 2016 Elsevier Inc. All rights reserved.