Cancer is the main target of research in the field of biology and pharmacology. Every year, thousands of cancer-related articles are published in specialized scientific journals. The development of new anticancer therapies is also one of the main interests of pharmaceutical companies. However, academia and big pharma have recently set their sights on cutting expenses related to drug development through co-operation protocols. Currently, various cellular viability assays (kits) are available in the market. However, most of these kits are expensive. To fulfill this need, there is a crescent demand for new protocols, such as cellular viability and cytotoxicity assays, that can be used for drug development in a faster and cheaper manner. In the present study, the application of Crystal Violet (CV), a compound that is relatively cheap, to determine the cellular viability of breast cancer and cervical cancer cells was evaluated. As no uniform and/or optimized CV cellular viability assay has been described in literature, the main focus of this work was the optimization procedure of a CV protocol, and its application for high-throughput screening (HTS) of new therapeutics. Two points of interest for the optimization of the protocol were considered: the possible fixation of cells and the concentration of the CV solution used for cell staining. The obtained results show there is an increase in absorbance proportional to the number of seeded cells for all CV protocol variations. The optimization procedure was successful, as it was shown that fixing and staining cells with a CV solution of higher concentration increased sensibility and decreased the variance of the signal/cell ratio in comparison with other tested protocols. It was also shown that the optimized CV assay may also be used as an alternative method for drug efficacy screening to other cellular viability assays widely described in literature, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Resazurin assays. It was also observed that the optimized CV assay may be applied for HTS of new anticancer drugs. Overall, as CV is a compound that is cheap to acquire, it may be used during anticancer drug development in the future.