Reactive Oxygen Species (ROS) are short-lived, highly reactive oxygen-containing molecules that can induce DNA damage and affect the DNA damage response (DDR). Pre-clinical and clinical evidence shows that ROS influence genotoxic stress caused by chemotherapeutic agents and ionizing radiation. Recent studies have provided insights into how ROS influence the cellular response to DNA damage, particularly in the context of Double Strand Breaks (DSBs). This has led to the clinical evaluation of agents modulating ROS in combination with genotoxic therapy for cancer, with mixed success. These studies indicate context-dependent outcomes, highlighting the need for additional pre-clinical research. The DDR is a network of events that includes DNA damage recognition, activation of checkpoints, cell cycle arrest, and repair-apoptosis and immune clearance. ROS can mediate DNA damage by generating free radicals, oxidizing nucleoside bases, and inducing mitochondrial DNA lesions. Oncogenic replication stress, a source of endogenous DNA damage, also involves ROS, which can affect replication fork progression and cause genomic instability. ROS can modulate the DDR by influencing sensor kinases like ATM and ATR, chromatin remodelers, and signal transduction pathways. They can also affect cell cycle progression and p53 transcriptional response, leading to cell death or resistance to chemotherapy and radiation. In clinical settings, ROS modulators have shown mixed efficacy in combination with chemotherapy and radiotherapy. Further research is needed to identify specific ROS species involved in DNA damage and DDR modulation, as well as to understand the dose-dependent effects of ROS on protein activity. The role of ROS in chemo/radioresistance and its potential as a therapeutic target requires further investigation.Reactive Oxygen Species (ROS) are short-lived, highly reactive oxygen-containing molecules that can induce DNA damage and affect the DNA damage response (DDR). Pre-clinical and clinical evidence shows that ROS influence genotoxic stress caused by chemotherapeutic agents and ionizing radiation. Recent studies have provided insights into how ROS influence the cellular response to DNA damage, particularly in the context of Double Strand Breaks (DSBs). This has led to the clinical evaluation of agents modulating ROS in combination with genotoxic therapy for cancer, with mixed success. These studies indicate context-dependent outcomes, highlighting the need for additional pre-clinical research. The DDR is a network of events that includes DNA damage recognition, activation of checkpoints, cell cycle arrest, and repair-apoptosis and immune clearance. ROS can mediate DNA damage by generating free radicals, oxidizing nucleoside bases, and inducing mitochondrial DNA lesions. Oncogenic replication stress, a source of endogenous DNA damage, also involves ROS, which can affect replication fork progression and cause genomic instability. ROS can modulate the DDR by influencing sensor kinases like ATM and ATR, chromatin remodelers, and signal transduction pathways. They can also affect cell cycle progression and p53 transcriptional response, leading to cell death or resistance to chemotherapy and radiation. In clinical settings, ROS modulators have shown mixed efficacy in combination with chemotherapy and radiotherapy. Further research is needed to identify specific ROS species involved in DNA damage and DDR modulation, as well as to understand the dose-dependent effects of ROS on protein activity. The role of ROS in chemo/radioresistance and its potential as a therapeutic target requires further investigation.