Dengue Virus (DNEV), West Nile Virus (WNV), Yellow Fever Virus (YFV), Japanese Encephalitis Virus (JEV) and Hepatitis C Virus (HCV) are members of flaviviridae family which infect millions of people worldwide. Developing drug against the said virus is urgently needed. Screening hundreds of natural and chemical compounds to find more competent anti viral drugs by using whole virus assay is uneconomic, time consuming and needs virology experts. The present invention disclosed a novel universal flavivirus protease assay kit comprising 5 types of recombinant flavivirus protease and specific fluorogenic peptide substrates to characterize the activity and kinetic parameter of the recombinant flavivirus protease. This novel universal flavivirus protease assay kit provides a convenient assay for high throughput screening of 5 types of flaviviruses protease inhibitors in a single kit.