Cell viability and proliferation assays are a fundamental tool in the drug discovery process. They are used to evaluate both the potency of compounds, as well as their toxicity profiles for drug safety assessments. Many commonly used plate reader assays (e.g. ATPluminescence measurements) assume a linear relationship between the assay signal, the number of cells and their viability. However, as this application note illustrates, this assumption is not always justified: ATP-luminescence measurements can significantly overestimate toxicity and underestimate potency, leading to false-negative viability/efficacy hits.