Granzymes are cell death-inducing serine proteases released from cytotoxic granules of cytotoxic T lymphocytes and natural killer cells during granule exocytosis in response to viral infection or against transformed cells marked for elimination. A critical cofactor for the granule exocytosis pathway is perforin, which mediates the entry of granzymes into target cells, where they cleave specific substrates that initiate DNA fragmentation and apoptosis. One of the biggest challenges in studying the biology of granzymes has been the functional redundancy of granzymes in animal models making an in vitro experimental system essential. This chapter discusses methods to study granzyme function in vitro under physiologically relevant experimental conditions.