The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro and mutational analysis confirmed the additional cleavage site in Hip. Since Hip's role in apoptosis is unknown, its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization, therefore it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer cell (NK) mediated death. Hip is cleaved at both GrB cleavage sites during NK mediated cell death in a caspase independent manner and its cleavage is due solely to GrB and not other granule components. Furthermore, Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T cell line, suggesting that Hip is a substrate unique to GrB. RNAi mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis, indicating that GrB's proteolysis of Hip contributes to death induction. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contributes to understanding the interplay between stress response and apoptosis.