A series of cyclic active-site directed inhibitors of the NS2B-NS3 proteases from Zika- (ZIKV), West Nile- (WNV), and Dengue-4 (DENV4) viruses was designed. The most potent compounds contain a reversely incorporated d-lysine residue in P1 position, where its side chain is connected to the P2 backbone, its a-amino group is converted into a guanidine for interaction with the conserved Asp129 side chain in the S1 pocket, and its carboxyl group is used for cyclization to the P3 lysine residue via an appropriate linker segment. The most potent compounds inhibit the ZIKV protease with K i values between 1.5 – 5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the design of the inhibitors. The binding mode of these compounds also explains their high selectivity as NS2B-NS3 inhibitors, which possess a negligible inhibitory effect on trypsin-like serine proteases or furin-like proprotein convertases. Although the WNV and DENV4 proteases are less efficiently inhibited, a comparable ranking of the potencies was observed for all three flavivirins suggesting that this compound type could serve as pan-flaviviral protease inhibitors.© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.