Aim. Vorinostat is ahistone deacetylase inhibitor approved for the treatment of acute T-cell lymphoma The recent studies have shown a significant potential of Vorinostat in the mono- and polytherapy of breast cancer as well. Administration of Vorinostat causes cell cycle arrest and apoptosis in neoplastic cells both in vivo and in vitro. Several analogues of Vorinostat were synthesized at Enamine Ltd. to improve the pharmacokinetics, selectivity and efficacy of the parental molecule. In this study, we aimed to test the effect of Vorinostat and its analogues on the viability of breast adenocarcinoma MCF-7 cell line using multiplexed High Content Imaging assay. Conclusions. High Content imaging assay coupled with luminescent cytotoxicity assessment was successfully optimized to investigate the potential cytotoxic and apoptotic anticancer effects of Vorinostat analogues in the breast cancer model, laying a foundation for further exploration of their therapeutic potential. We identified optimal conditions for the cell growth and confluency, as well as concentrations for staining solution components and reference compounds.