Loss-of-function mutations within the retinoblastoma gene RB1 are typical in a number of treatment-refractory cancers for example small-cell cancer of the lung and triple-negative cancer of the breast. To recognize drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors utilizing a cancer cell panel profiling approach enhanced to discern cytotoxic from cytostatic effects. Inhibitors from the Aurora kinases AURKA and AURKB demonstrated the most powerful RB1 association within this assay. LY3295668, an AURKA inhibitor with more than 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and results in durable regression of RB1 mut tumor xenografts at exposures which are well tolerated in rodents. Genetic suppression screens identified enforcers from the spindle-set up checkpoint (SAC) essential for LY3295668 cytotoxicity in RB1-deficient cancers and advise a model where a primed SAC results in a unique reliance upon AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of the synthetic lethal interaction between RB1 and AURKA inhibition, and also the discovery of the drug that may be dosed continuously to attain uninterrupted inhibition of AURKA kinase activity without myelosuppression, advise a new approach to treat RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This information is highlighted within the Within This Issue feature, p. 151.