The urokinase receptor (uPAR) is a cell surface receptor that binds to the serine protease urokinase-type plasminogen activator (uPA) with high affinity. This interaction is beneficial for extravascular fibrin clearance, but it has also been associated with a broad range of pathological conditions including cancer, atherosclerosis, and kidney disease. Here, starting with a small molecule that we previously discovered by virtual screening and cheminformatics analysis, we design and synthesize several derivatives that were tested for binding and inhibition of the uPAR . uPA interaction. To confirm the binding site and establish a binding mode of the compounds, we carried out biophysical studies using uPAR mutants, among them uPAR(H47C-N259C), a mutant previously developed to mimic the structure of uPA-bound uPAR. Remarkably, a substantial increase in potency is observed for inhibition of uPAR(H47C-N259C) binding to uPA compared to wild-type uPAR, consistent with our use of the structure of uPAR in its uPA-bound state to design small-molecule uPAR . uPA antagonists. Combined with the biophysical studies, molecular docking followed by extensive explicit-solvent molecular dynamics simulations and MM-GBSA free energy calculations yielded the most favorable binding pose of the compound. Collectively, these results suggest that potent inhibition of uPAR binding to uPA with small molecules will likely only be achieved by developing small molecules that exhibit high-affinity to solution apo structures of uPAR, rather than uPA-bound structures of the receptor.