The activity of dengue NS2B-NS3pro to cleave the fluorogenic peptide substrate at different temperature conditions. The enzyme kinetic assay was carried out to evaluate dengue NS2B-NS3pro activity at temperature of mosquitoes (28°C), humans (37°C) and humans with severe dengue fever (40°C). Serial concentrations of the substrate peptide were used to assess the activity of recombinant DENV-2 protease. Triplicates were performed for all experiments and the relative fluorescence units (rfu) were measured using Tecan Infinite M200 Pro fluorescence spectrophotometer. Substrate cleavage was optimized at the emission at 440 nm upon excitation at 350 nm. After normalization of the results with the substrate incubated at same conditions, the highest activity of recombinant NS2B-NS3pro was observed at 28°C and lowest activity was at 40°C.