Primary human hepatocyte (PHH) suspension assays are widely used to evaluate drug metabolism and drug–drug interactions; however, their short viability (4–6 hours) limits their applicability for low-clearance compounds. While adherent and co-culture systems extend culture duration, they often introduce complexity and may not fully support long-term metabolic function. Here, we present a hepatocyte-only 3D model that supports sustained PHH culture for over three weeks through spheroid culture, optimized donor selection and ultra-low attachment plate formats. These spheroids maintain stable morphology, viability, and metabolic enzyme activity for several weeks, enabling extended evaluation of low-clearance compounds for over 10 days. Validation using reference compounds demonstrated accurate half-life predictions consistent with published in vitro data. Compared to conventional 2D systems, this 3D spheroid platform better preserves in vivo-like liver architecture and function, thus providing enhanced physiological relevance. Pre-qualification of hepatocyte lots further ensures reproducibility and scalability in a 96-well high-throughput format. This system represents a robust and streamlined approach for drug metabolism, drug–drug interactions, and hepatotoxicity studies.
