Histone deacetylase (HDAC) inhibitors represent a novel class of therapeutic agents for cancer treatment. These enzymes play a pivotal regulatory role on chromatin epigenetics by deacetylation of histone proteins and lead to the production of reactive oxygen species (ROS) via activation of apoptotic pathway and autophagy. Surprisingly, the HDAC inhibitor-mediated ROS production in various cancer cells such as leukemia was found to be elevated by a combination of DNA damaging agents and proteasome inhibitors. In contrast, cancer cells possess proteins such as Trx and Trx reductase, as well as GSH peroxidase (Gpx) that help in preventing the oxidative stress of the cells by reducing the H₂O₂. Various bioinformatics tools can be utilized to understand the HDAC inhibitor-mediated differential gene expression data obtained by using the Affymetrix platform as well as the Illumina platform. Further, Gene Ontology (GO) and pathway analyses tools reveal pro-apoptotic gene expression signature and intrinsic apoptotic pathway during ROS generation. The differentially expressed genes were further subjected to Ingenuity Pathway Analysis (IPA) tool to study the associations of various molecular and cellular functions. ReMap (regulatory map of TF binding sites) analysis tool demonstrated the chromatin occupancy by proteins such as bromodomain-containing protein-4 (BRD4) and MYC proteins at their binding sites during HDAC inhibitor-treated cancer cells. Studies on HDAC inhibitor-mediated ROS generation and the tumor-suppressive effects can be better studied by using a combination of various molecular and bioinformatics methods that would help in better therapy against cancer.