Ac-EVKKQR-pNA is a competitive chromogenic para-nitroanilide substrate corresponding to the P6-P1 segment amino-terminal to the NS2B-NS3 cleavage site but with a more reactive, hydrolytically cleavable, para-nitroanilide at the P1’ position. Ac-EVKKQR-pNA is promising for research of dengue 2 virus and flavivirus virus infection.