Although the mosquito-borne Zika virus was discovered in the late 1940s of the 20th century, for years it was neglected, as the disease in humans was rare and relatively mild. Viral NS2B-NS3 protease is essential for virus replication, and except for maturation of viral proteins, it also modulates the infection microenvironment to facilitate virus invasion. Here, we report the combinatorial chemistry approach for the synthesis of internally quenched substrates of the Zika virus NS2B-NS3 protease that were optimized in prime positions of the peptide chain. Final substrate ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 displays an excellent kinetic parameter (k cat/K M reaching nearly 1.26 × 108 M-1 × s-1), which is over 10 times greater than previously reported (7.7 × 106 M-1 × s-1) substrate. Moreover, it was found to be selective over West Nile virus protease.