Caspases are fundamental to many essential biological processes, including apoptosis, differentiation, and inflammation. Unregulated caspase activity is also implicated in the development and progression of several diseases, such as cancer, neurodegenerative disorders, and sepsis. Unfortunately, it is difficult to determine exactly which caspase(s) of the 11 isoforms that humans express is responsible for specific biological functions. This lack of resolution is primarily due to highly homologous active sites and overlapping substrates. Currently available peptide-based inhibitors and probes are based on specificity garnered from peptide substrate libraries. For example, the canonical tetrapeptide LETD was discovered as the canonical sequence that is optimally recognized by caspase-8; however, LETD-based inhibitors and substrates promiscuously bind to other isoforms with equal affinity, including caspases-3, -6, and -9. In order to mitigate this problem, we report the identification of a new series of compounds that are >100-fold selective for inhibiting the initiator caspases-8 and -9 over the executioner caspases-3, -6, and -7.C aspases are a family of cysteine-dependent aspartatedirected proteases with 11 human isoforms that are traditionally known for their indispensible roles in the initiation (caspases-2, -8, -9, -10) and execution (caspases-3, -6, -7) of apoptosis. 1 Other family members, including caspases-1, -4, and -5, are important regulators of inflammation and induce pyroptotic cell death as a result of microbial infection. 2 Intriguingly, recent reports implicate caspase activity as being critical for a variety of other essential biological processes, such as DNA repair signaling and tumor suppression, 3 skeletal muscle differentiation, 4 B-cell proliferation, 5 dendritic pruning and neuronal plasticity, 6 embryonic stem cell differentiation through Nanog cleavage, 7 nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) activation, 8 lymphocyte and monocyte differentiation and development, 9 and keratinocyte differentiation and skin barrier formation. 10 The utility of caspase-dependent proteolysis in an array of cellular functions is continually expanding, and additional caspase roles will likely be discovered with the development of highly potent and specific chemical inhibitors and probes.Active caspases have many significant irreversible consequences and as such are stored as inactive proenzymes, or procaspases, inside of the cell. 11 Intriguingly, several small molecules have been identified in high-throughput screens that promote executioner procaspase maturation using in vitro and in vivo models. 12−14 Aberrant caspase activity is implicated in the development and progression of several diseases, such as neurodegenerative disorders, 15 cancer, 16 cardiovascular disease, 17 and sepsis. 18 Moreover, caspases represent potential drug discovery targets for the treatment of these diseases, and it is therefore imperative to elucidate the exact biological roles of each i...