Tumor acidosis is associated with increased invasiveness and drug resistance. Here, we take an unbiased approach to identify vulnerabilities of acid-exposed cancer cells by combining pH-dependent flow cytometry cell sorting from 3D colorectal tumor spheroids and transcriptomic profiling. Besides metabolic rewiring, we identify an increase in tetraploid cell frequency and DNA damage response as consistent hallmarks of acid-exposed cancer cells, supported by the activation of ATM and ATR signaling pathways. We find that regardless of the cell replication error status, both ATM and ATR inhibitors exert preferential growth inhibitory effects on acid-exposed cancer cells. The efficacy of a combination of these drugs with 5-FU is further documented in 3D spheroids as well as in patient-derived colorectal tumor organoids. These data position tumor acidosis as a revelator of the therapeutic potential of DNA repair blockers and as an attractive clinical biomarker to predict the response to a combination with chemotherapy. Synopsis pHLIP-based sorting of acid-exposed cancer cells from 3D spheroids identifies tetraploidy and DDR as hallmarks of tumor acidosis. Drugs targeting ATM and ATR preferentially inhibit growth of acid-exposed cancer cells and sensitize them to chemotherapy. Acid-exposed cancer cells undergo tetraploidy and trigger a DNA Damage Response (DDR). Both ATM and ATR pathways are activated in response to acidic pH e . Growth of acid-exposed cancer cells is hampered by ATM and ATR inhibitors. DDR inhibitors sensitize resistant acid-exposed cancer cells to 5-FU. pHLIP-based sorting of acid-exposed cancer cells from 3D spheroids identifies tetraploidy and DDR as hallmarks of tumor acidosis. Drugs targeting ATM and ATR preferentially inhibit growth of acid-exposed cancer cells and sensitize them to chemotherapy.