Background/Objectives: Cancer persists as a leading concern in the current medical field, and current therapies are limited by toxicity, cost, and resistance. Targeted inhibition of tubulin polymerization is considered as a promising therapeutic strategy for cancer treatment. Methods: Thirty-one new tubulin polymerization inhibitors were designed via molecular hybridization techniques, and BLI technology was employed to quantitatively investigate their interactions with tubulin. Antiproliferative activities against MCF-7, MDA-MB-231, A549, and HeLa cell lines was evaluated using the CCK8 assay. Apoptosis induction and cell cycle arrest were analyzed by flow cytometry. The anti-tumor activity of compound B6 was validated in a mouse melanoma tumor model. Results: Compounds exhibited varying degrees of antiproliferative activity against four tumor cell lines. Among them, compound B6 was the most promising candidate and displayed strong broad-spectrum anticancer activity with an average IC₅₀ value of 2 μM. The mechanism studies revealed that compound B6 inhibited tubulin polymerization in vitro, disrupted cell microtubule networks, and arrested the cell cycle at G2/M phase. Furthermore, B6 displayed significant in vivo antitumor efficacy in a melanoma tumor model with tumor growth inhibition rates of 70.21% (50 mg/kg). Conclusions: This work shows that B6 is a promising lead compound deserving further investigation as a potential anticancer agent.