After the US FDA′s approval of brentuximab vedotin in 2011 and ado-trastuzumab emtansine in 2013, the field of antibody-drug conjugates (ADCs) has gained tremendous momentum, and significant research efforts are directed to identify more effective payloads and to achieve more efficacy for higher safety profile. There are over 30 ADCs currently in clin. evaluation. The majority of ADCs in clin. evaluation utilize the highly potent tubulin-interacting agents, maytansinoids or auristatins. A few ADCs in clinics have incorporated other potent effector mols., such as the topoisomerase 1 inhibitor SN-38 or the DNA interacting agents calicheamicin and pyrrolobenzodiazepines. The tetrapeptide tubulysins which have been reported the exceptional potency exceeding that of vinblastine, taxol and epothilones are interesting cytotoxic agents for ADC studies. Here we designed and synthesized Tubulysin D analogs bearing conjugatable linkers at their C-terminus (tubutyrosine (Tut)). In vitro, these free conjugatable Tubulysin D analogs are much less potent than a maytansinoid, DM1SMe, due to the hydrophilicity of these angents. Once conjugated to an antibody, the conjugates of these cytotoxic agents were much highly cytotoxic and enhanced immunol. specific cell killing at subsaturating doses. In vivo, ADCs bearing these cytotoxic agents demonstrated better antitumor activities than existing ADC drugs in several xenografttumor models.