This protocol outlines the use of flow cytometry to assess key cellular parameters in 4T1 mouse breast cancer cells following treatment with various compounds. The aim is to evaluate membrane integrity, reactive oxygen species (ROS) levels, and cell cycle progression to understand the cellular effects of oxidative stress and chemotherapeutic agents. Membrane integrity is assessed using propidium iodide (PI) staining, ROS levels are measured with the H2DCFDA probe, and cell cycle distribution, including the Sub-G1 population (apoptotic cells), is analyzed with PI and RNase A treatment. The cells are treated with phytocompound, dacarbazine, and hydrogen peroxide to induce oxidative stress, and the resulting changes in cell viability, ROS production, and cell cycle dynamics are measured by flow cytometry. This protocol provides a comprehensive approach to studying the molecular mechanisms of drug-induced cell death and oxidative stress in cancer cells, using flow cytometry as a quantitative tool for cellular analysis.