Cytolytic enzymes (CEs) are critical mediators of anti-viral and -tumor immunity; however, as a number of molecules belong to this enzyme family, our understanding of CEs remains limited. Specifically, it remains unclear what combinations of granzymes and perforin (Perf) are expressed by various immune cells and how CE content relates to cellular differentiation. Using polychromatic flow cytometry, we simultaneously measured expression of the most common human CEs [granzyme A (gA), granzyme B (gB), and Perf] alongside markers of alphabeta and gammadelta T cell maturation (CD45RO, CCR7, CD27, CD57). Additionally, we measured CE content in NK cell subsets (defined by their expression of CD16 and CD56). We found that among a wide variety of immune cells, CE content was linked to cellular maturity. Moreover, common expression patterns were shared across cell types, such that gB+ cells always contained gA, and Perf+ cells were primarily gA+ gB+. Most importantly, CD57 expression correlated strongly with simultaneous expression of gA, gB, and Perf. Thus, the use of CD57 provides a means to easily isolate viable cells with high cytolytic potential, without the need for lethal fixation/permeabilization techniques.