Enterovirus-D68 has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Despite previous attempts, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. However, the structural characterization of EV68- 3C protease bound to its substrates, and therefore the substrate specificity has been lacking. For other viral proteases, we previously demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. A better understanding of this molecular basis of drug resistance is therefore central to developing robust protease inhibitors with a high barrier to drug resistant mutations. As a result of this need, my research characterizes the structural basis of specificity of EV68-3C protease and explores the resistance profiles of current inhibitors with plans for developing robust protease inhibitors. Chapter II explores the structural characterization EV68-3C Protease, both with computational modeling and experimental protein crystallography, to define the common volume shared by EV68-3C protease viral substrates – the substrate envelope. We utilize the substrate envelope to assess potential sites of resistance as well as uncover protein conformational changes during ligand-binding. Chapter III assesses additional 3C and 3C-like inhibitors through protein crystallography to further understand possible resistance mutations where inhibitors protrude from the substrate envelope. We also analyzed structural patterns and motifs from ligand-bound protein co-crystals that can aid in the design of robust EV68-3C inhibitors. I hope this works lays down a strong foundation for other scientists to expand upon when developing robust therapeutics against this debilitating pathogen as well as applying this knowledge to similar viruses.