Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy. More than half of DLBCL patients achieve long-term remission after treatment, but a third relapse after conventional Rituximab (R)-based chemotherapy regimens, such as CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone). Cancer cells are exposed to chronic replication stress, which impedes the duplication of their genome. Functional DNA repair pathways are therefore important for the survival of cancer cells. This dependence can be exploited therapeutically to hamper repair of the intrinsic DNA damage occurring during replication or to exacerbate DNA damage induced by chemotherapy. Using CRISPR-Cas9 screening, we identified CHEK1, WEE1, ATR and RAD51 DNA repair factors as essential genes in DLBCL cells. According to these results, we investigated the effect of small molecules targeting DNA replication stress and DNA repair mechanisms, alone or in combination with the R-CHOP genotoxic agents, cyclophosphamide and doxorubicin. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone, a total of 3 synthetic lethal combinations have been identified including cyclophosphamide with CHK1/2 inhibitor, cyclophosphamide and ATR inhibitor and doxorubicin with DNAPK inhibitor. Co-treatment with these molecules led to cell death, DNA damage induction and cell cycle arrest in DLBCL cells more efficiently than genotoxic agents alone. These data have been validated using primary DLBCL cells from patients. Our results open new perspectives for therapeutic approaches exploiting the synthetic lethality of genotoxic agents with DNA repair inhibitors to improve the therapeutic outcome of patients with DLBCL.