Tubulin binding agents are a potent group of cancer chemotherapeutics. Most of these substances are naturally derived compounds. A novel substance class of destabilizing agents is the group of tubulysins. The tubulysins and their derivative pretubulysin have shown high efficacy in vitro and in vivo. Due to their complex chemical structures, one major bottleneck of the tubulysins is their accessibility. Biotechnological as well as chemical production is challenging, especially on larger scales. Thus, the synthesis of chemically simplified structures is needed with retained or improved biological activity. Herein is presented the biological evaluation of two pretubulysin derivatives [2-desmethylpretubulysin AU816 (1) and phenylpretubulysin JB337 (2)] in comparison to pretubulysin. Both 1 and 2 display a simplification in chemical synthesis. It was shown that both compounds exhibited potent biological activity against cancer cells. These simplified compounds inhibited tubulin polymerization in the nanomolar range. The cytotoxic effects of 1 and 2 were in a similar range, when compared with pretubulysin [IC50 (nM): pretubulysin: 0.6; 1: 10; 2: 100]. Furthermore, it was shown that cell cycle arrest is induced and migration is hampered in MDA-MB-231 breast cancer cells. In conclusion, 1 was shown to be about 10-fold more active than 2 and as potent as pretubulysin.