Abstract High-content microscopy offers a scalable approach to screen against multiple targets in a single pass. Prior work has focused on methods to select “optimal” cellular readouts in microscopy screens. However, methods to select optimal cell line models have garnered much less attention. Here, we provide a roadmap for how to select the cell line or lines that are best suited to identify bioactive compounds and their mechanism of action (MOA). We test our approach on compounds targeting cancer-relevant pathways, ranking cell lines in two tasks: detecting compound activity (“phenoactivity”) and grouping compounds with similar MOA by similar phenotype (“phenosimilarity”). Evaluating six cell lines across 3214 well-annotated compounds, we show that optimal cell line selection depends on both the task of interest (e.g. detecting phenoactivity vs. inferring phenosimilarity) and distribution of MOAs within the compound library. Given a task of interest and set of compounds, we provide a systematic framework for choosing optimal cell line(s). Our framework can be used to reduce the number of cell lines required to identify hits within a compound library and help accelerate the pace of early drug discovery.