The datasets provided are associated with the manuscript titled "High-throughput Screening Data Generation, Scoring, and FAIRification: a case study on nanomaterials." These datasets include in-vitro images and cell viability data from five distinct assays: CellTiter-Glo, DAPI, γH2AX, 8OHG, and Caspase. A specially developed and filled HTS_METADATA templates are included to ensure a harmonized reporting format, facilitating the description, reading, and annotation of the raw data. The manuscript utilizes two datasets for software testing: one from the caLIBRAte project and another involving quantum dots from the HARMLESS project. The caLIBRAte raw data is provided in CSV and TXT formats. The quantum dots data, both raw and preprocessed, are available in Nexus format, which can be visualized at https://h5web.ideaconsult.net/.