The drug case studies provided in Chapter 12 make a case for at least considering 3D cell-based biosensing or bioassay in HTS. Despite these cases, a skeptical director of a drug discovery program from one of the major pharmaceutical companies once challenged this author after presenting these cases as a basis for the merits of adopting 3D cultures in drug discovery. “Show me a couple of drugs that were promising when tested with 2D cultures but failed after approval and 3D cultures testing after withdrawal reveals the drugs as nonpromising; and I will be totally convinced,” the challenger said. A thorough search of the literature did not yield any results that satis ed the condition the challenger put forth. The presence of such data is considered critical to the narrative toward persuading pharmaceutical, biopharmaceutical, and biotechnological companies to deploy 3D biosensor technology in their drug discovery programs. Such a study is proposed herein. The execution and success of such a study will provide answers to the challenge and probably accelerate the adoption of 3D systems in drug discovery programs. The proposal focuses on hepatotoxicity with hepatocyte-like cells derived from human embryonic stem cells (hESCs) and drugs that were approved but later withdrawn. To be consistent with HTS applicability of the resultant knowledge, the study should be conducted in three commercially available 3D high-density (e.g., 96-and 1536-well palates) with a 2D high-density plate as control. The three subhypotheses driving the study are as follows: (1) stem cell-derived hepatocyte-like cells cultured in 3D microenvironments emulate the in vivo liver cells better than their 2D counterparts, (2) in comparison to 2D, 3D cell-based biosensors improve the hepatotoxicity predictability of experimental drugs, and (3) mitochondrial inner transmembrane potential (Δψm) signal from 3D cells, in particular stem cell-derived hepatocyte-like cells, is easily distinguishable from background noise and can be used to yield a positive Z′ factor in the proposed 3D plates.