In the scenario of drug discovery, numerous in vitro testing initiatives had been established. Thus far, no general methodology is reputable and literature on this hot topic is scarce. In this respect, we propose a strategy based on a Phenotypic Drug Discovery approach. Within our program directed at the discovery of new antitumor agents, we have focused our attention on compounds that disturb the cell cycle. Our strategy relies on the use of a set of biological assays organized in a modular fashion. Herein, we exemplified this strategy with a family of propargylic enol ether derivatives. Using different assays in sequential stages and in a stepwise manner, our studies allowed us to understand the bioactivity of this family of compounds and led us to identify tubulin as the main molecular target.