To determine the inhibitory activities of flavonoids against NS2B-NS3 protease of ZIKA virus (ZIKV NS2B-NS3(pro)) expressed in Escherichia coli BL21 (DE3) and their structure activity relationship. ZIKV NS2B-NS3(pro) was expressed in E. coli BL21(DE3) as a 35 kDa protein. It had a K (m) of 26 A mu M with the fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edan. The purified ZIKV NS2B-NS3(pro) was used for inhibition and kinetic assays to determine the activities of 22 polyphenol compounds. These polyphenol compounds at 100 A mu M inhibited the activity of ZIKV NS2B-NS3(pro) by 6.2-88%. Seven polyphenol compounds had IC50 ranging from 22 +/- 0.2 to 112 +/- 5.5 A mu M. Myricetin showed a mixed type inhibitory pattern against ZIKV NS2B-NS3(pro) protease. Its IC50 value was 22 +/- 0.2 A mu M with a K (i) value of 8.9 +/- 1.9 A mu M. The chemical structure of a polyphenol compound and its inhibitory activity against ZIKV NS2B-NS3(pro) can be explored to develop highly selective inhibitors against ZIKV NS2B-NS3(pro).