Investigation of Granzyme B (GrB) function and pathophysiology in both human settings and rodent models increasingly involve the use of indirect immunofluorescence imaging and fluorescence-activated cell sorting, which requires reliable GrB antibodies that do not recognise other closely related granzymes. Here, we describe the validation (using a set of recombinant granzymes, and GrB-deficient cells) and application of widely available monoclonal antibodies to specifically monitor GrB in human or mouse cells.