Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members.