Studying the mechanism of action for a small molecule is always challenging, not only because of the complex molecular interactions involved, but also due to the intricacies of intracellular processes, the diverse response of different cell types, and the evolving nature of biological systems under the influence of these compounds. Despite these challenges, it remains an indispensable task as it provides fundamental insights into cellular function, drug action, and can pave the way for the development of more effective therapeutic strategies. This thesis uses largazole, a class I HDACs inhibitor, as an example to show how to study the mechanism of action for a small molecule from scratch using various modern techniques and tools. In Chapter 1, the background knowledge of histone deacetylase (HDAC), HDAC inhibitors (HDACi) and largazole are overviewed. In Chapter 2, we identify the targets of largazole using CRISPRa-HTP, validating these targets through parallel and orthogonal methods. Subsequently, we delve deeper into the relationship between largazole’s HDACi property and PRDX6, one of the identified targets. In Chapter 3, mechanism of action for PRDX6 knockdown induced sensitization of largazole was studied with the combination of ATAC-seq and ChIP-seq. And adaptive resistance of largazole was investigated using scRNA-seq. iii In Chapter 4, direct interaction between largazole and PRDX6, SP1 was studied using microscale thermophoresis, intact mass spectrometry and functional assays. In Chapter 5, cell type specificity and drug specificity were studied using various cell types and HDACi. Differential action was observed between largazole and paragazole. In Chapter 6, mechanism of action was studied in an animal model and PRDX6 as a biomarker for cancer aggressiveness was proposed. In Chapter 7, conclusions and summaries of the current works are presented. In addition, perspectives and future directions are displayed