Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. Infact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.