Caenorhabditis elegans genome has four genes (ced-3, csp-1, csp-2, and csp-3) encoding caspase-like proteins. Among these four proteins, CED-3 is the most well-known cell-killing caspase. Elucidation of the role of CED-3 as a central component of the apoptotic pathway in C. elegans has contributed to the understanding of the more complex apoptosis network in mammals and in other metazoa. In the highly conserved pathway of programmed cell death in C. elegans, CED-3 functions at the terminal step of this cell-killing pathway. Identification of CED-3 caspase substrates is essential for bridging the gaps between CED-3 activation and various downstream cell death execution events. If a protein is cleaved by CED-3 in vitro, this protein could be a potential CED-3 substrate in vivo. Here, we describe the method for purification of active CED-3 caspase. We will also describe in vitro assays for determining CED-3 proteolytic activity, CED-3 substrates, and CED-3 cleavage sites in the substrates.