Antimitotics, namely microtubule-targeting agents (MTAs) such as paclitaxel, are successful chemotherapeutics in use for cancer therapy. However, the associated toxicity and resistance have led to the emergence of the second-generation antimitotics (SGAs) exhibiting promising antitumor activity in preclinical assays. Unfortunately, SGAs failed in monotherapy clinical trials. One reason is that two-dimensional (2D) culture lacks cell-cell contacts and natural tumor microenvironment, important in tumor signaling and drug response. Consequently, three-dimensional (3D) spheroid cultures emerged as promising tool to fill this gap, being able to recapitulate many characteristics of tumors microenvironment. Here, we propose i) to understand why SGAs failed in monotherapy clinical trials; and ii) to test a strategy for increasing their efficacy against cancer, so as to give SGAs a second opportunity and to facilitate the design of successful human therapy trials.