The CellTiter-Glo 3D Cell Viability Assay is a homogeneous method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells. This ready-to-use reagent is based on the original CellTiter-Glo Luminescent Cell Viability Assay chemistry and eliminates the need to combine buffer with lyophilized substrate when preparing reagent. The CellTiter-Glo 3D Cell Viability Assay is formulated with more robust lytic capacity and is designed for use with microtissues produced in 3D cell culture. The homogeneous assay procedure involves addition of a single reagent (CellTiter-Glo 3D Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required. This assay is compatible with multiwell-plate formats, making it ideal for automated high-throughput screening (HTS). The CellTiter-Glo 3D Assay has been used successfully with 3D microtissue cell culture produced via hanging-drop plates, ultra-low attachment plates, Matrigel-coated plates, agarose-coated plates, cultures suspended in methylcellulose and Alvetex plates.