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Summary: Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.