Apoptosis executed by the mammalian caspase family plays a fundamental role in cellular homeostasis. Deregulation of this process is associated with several human diseases. The multimerization of ligand-induced death receptors results in the recruitment of the death inducing signaling complex and autocatalytic activation of initiator caspases, including caspase-8 and -10. However, it is still unclear how initiator caspases trigger and control the early apoptotic signaling pathways, partly because the downstream proteolytic cleavage targets of the initiator caspases are not completely known. Although it is known that a number of proteins are cleaved by various members of the caspase family, the identification of specific cleavage substrates of the initiator caspases 8 and 10, has been hindered by a lack of systematic and broadly applicable strategies for substrate identification. In the present study we constructed a mouse cDNA library and used it to perform a systematic, genome-wide screen for novel in vitro substrates of caspase-8 and -10. From this, we successfully identified six putative caspase substrates, including five novel proteins (ABCF1, AKAP1, CPE, DOPEY1 and GOPC1) that may be targeted specifically by the initiator caspases 8 and 10 during the early stages of apoptosis. These findings may provide useful information for elucidating the apoptotic signaling pathways downstream of the death receptors.