The gene encoding the dihydropteroate synthase (DHPS) of Streptococcus pneumonia has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified and utilised to develop a novel microplate assay for the detection of sulfonamides. The assay was based on the competition between sulfonamides and horse radish peroxidase (HRP) labeled sulfonamide derivative, 4-(4-aminobenzenesulfonylamino) benzoic acid (CS), for the immobilized protein. Under optimized conditions, 9 sulfonamides and para-aminobenzoic acid (PABA) could be detected below 100 ng/ml and 28 sulfonamides used in the study could be detected with IC50 values ranging from 426 to 50000 ng/ml. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of sulfonamides.