Upcyte® hepatocyte spheroids were grown simultaneously in Gri3D (SUN bioscience) and U-bottom ultra-low attachment 96WP to compare ease of use, throughput, assay sensitivity, and scalability for multi-assays. After exposure, drug effects on viability and enzymatic activities were assessed using Promega assays (CellTiterGlo® 3D, CellToxTM Green, LDH-GloTM, and P450-GloTM) and Tecan’s Spark Cyto multi-mode imaging plate reader. Readouts encompassed fluorescence and luminescence intensity measurements, along with imaging. The assays were performed directly on-plate, ensuring the micro tissues remain undisturbed. The Fluent Automation Workstation played a key role in streamlining the workflow, enabling precise and fast cell seeding and media exchanges. Our semi-automated 3D multiplexing approach, combining an innovative microwell plate, compatible cellbased kits, a multimode plate reader, and a liquid handler, provides valuable insights into drug impact on CYP
activity and microtissue viability, all within a single experiment. This innovative method advances our understanding of liver spheroid responses to drug exposure and holds promise for larger-scale compound assessments and drug screening using 3D cell cultures.