The serine protease granzyme B (GrB) is a key effector molecule in cell-mediated immunity, released by cytotoxic lymphocytes (CLs) to induce cell death in neoplastic or virus-infected cells. The ability to detect and measure GrB activity is important for understanding CLs. Unfortunately, such analyses are complicated by significant differences in the substrate specificities of human and mouse GrB, which is reflected by their different activities on commonly used peptide substrates. Here, we present methods for the detection of active human and mouse GrB in extracts from primary cells, and evaluate the sensitivity of the various substrates and inhibitors. Mouse splenocytes produce approximately 120-fold more GrB than similarly activated human cells, which allows the use of the hGrB substrate IETD-AFC to follow mouse GrB activity despite its unfavourable kinetic properties.