Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/β interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations. © 2021 American Chemical Society.