This work details the inhibition of BACE1, BACE2, and SARS-CoV-2 3CLpro through several novel and potent protease inhibitors. Nanomolar potency of BACE1 and BACE2 is achieved with all tested inhibitors, and the S2 subsite has been identified as a BACE2 selectivity determinant. This is supported by the observation that the novel BACE2 mutant, BACE2 L246N, displays increased potency and selectivity over BACE1 against this peptidomimetic scaffold. Nanomolar to micromolar potency of SARS-CoV-2 3CLpro is achieved with the compounds tested in this study. Kinetic data illustrates the allowed substitutions at the P1’, P1, P2, and P4 positions on two scaffolds: ML188 and GC376. Finally, this work presents the high-resolution crystal structures of four inhibitors bound to BACE1 and 12 inhibitors bound to SARS-CoV-2 3CLpro. These structural data help to explain the selectivity determinants of BACE1 and BACE2 and further enable structure-based drug design against these two enzymes for the treatments of Alzheimer's Disease and Type II Diabetes, respectively. Additionally, these structural data illustrate the flexibility of the GC376 scaffold at the P3/P4 position, providing a structural rationale for the observed differences in potency across the different analogs. These structural data further enable structure-based drug design against SARS-CoV-2 3CLpro for the treatment of COVID-19.