The synthesis of diverse three-dimensional libraries has become of paramount importance for obtaining better leads for drug discovery. Such libraries are predicted to fare better than traditional compound collections in phenotypic screens and against difficult targets. Herein we report the diversity-oriented synthesis of a compound library using rhodium carbenoid chemistry to access structurally diverse three-dimensional molecules and show that they access biologically relevant areas of chemical space using cheminformatic analysis. High-content screening of this library for antimitotic activity followed by chemical modification identified 'Dosabulin', which causes mitotic arrest and cancer cell death by apoptosis. Its mechanism of action is determined to be microtubule depolymerization, and the compound is shown to not significantly affect vinblastine binding to tubulin; however, experiments suggest binding to a site vicinal or allosteric to Colchicine. This work validates the combination of diversity-oriented synthesis and phenotypic screening as a strategy for the discovery of biologically relevant chemical entities.