Therapeutic proteins with specific effector functions play an increasingly important role in drug therapy. For example, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) predominantly kills cancer cells, while sparing normal cells. Here, we report the use of a secreted version of TRAIL as a therapeutic protein that induces apoptosis and kills surrounding cells in vivo, thus resulting in the dramatic reduction of glioma burden in mouse tumor models. Using a caspase-3-activatable aminoluciferin, we were able to show the induction of apoptosis specifically in S-TRAIL vector-infected gliomas. We also show that S-TRAIL-mediated apoptosis and resulting changes in tumor burden can be imaged in the same animal by dual-substrate bioluminescence imaging. The use of S-TRAIL as a therapeutic protein and the ability to image noninvasively both apoptosis and any other cellular events in real time have important clinical implications.