Maintaining genomic integrity is essential for cellular health and the prevention of genetic disorders, including cancer. When DNA damage is left unrepaired, it can lead to mutations that disrupt key cellular processes and promote disease progression. This chapter outlines a comprehensive protocol for assessing DNA damage across different stages of the cell cycle using flow cytometry—a powerful technique for quantitatively analyzing both cell cycle distribution and DNA damage markers. Key phases of the cell cycle (G1, S, G2, and M) are examined, with emphasis on DNA damage checkpoints and the repair mechanisms that preserve genomic stability. Cells utilize multiple repair pathways, including direct reversal, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double-strand break repair (DSBR). The protocol includes detailed steps for cell synchronization, DNA damage induction using agents such as UV light or hydrogen peroxide, and flow cytometric analysis following propidium iodide (PI) staining. Step-by-step procedures for cell culture, synchronization, fixation, staining, and data interpretation are provided. By enabling precise quantification of DNA damage and detection of cell cycle arrest, this method advances our understanding of DNA repair dynamics and genomic stability—offering critical applications in cancer research, genetic disease studies, and the development of targeted therapies and diagnostic tools.