AbstractWe describe the characterization of antigen binding fragments (Fab)–drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross‐linkers, are a class of payloads used in antibody–drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug‐to‐Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti‐HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti‐HER2 trastuzumab Fab‐rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen‐dependent cancer cell‐killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab–pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.