Purpose <p>Three-dimensional liver spheroids provide physiologically relevant models for studying hepatocyte function and drug-induced liver injury, yet inconsistencies in formation methods hinder reproducibility across laboratories.</p> Methods <p>To address this, we systematically compared HepG2 spheroid culture approaches—35 and 96 microwell agarose gels, microwell and single-well ultra-low attachment (ULA) plates, and hanging drop—across a range of seeding densities and culture durations.</p> Results <p>Morphological and functional analyses revealed that spheroid geometry, which governs nutrient and oxygen diffusion, strongly influenced hepatocyte-like function: 35-microwell agarose gels produced the most circular spheroids and exhibited significantly higher albumin secretion and ATP production than plate-based methods. However, no method fully combined repeatability, throughput, and functional robustness; instead, each platform presented distinct advantages and limitations in different areas.</p> Conclusion <p>An optimal method depends on application-specific priorities (e.g. throughput, cost, or physiological function). This comparative analysis establishes a quantitative framework for optimizing spheroid culture method selection and underscores the need for hybrid approaches that integrate the scalability of plate-based methods with the functional performance of agarose-based systems. By improving the reproducibility of in vitro hepatic models, this work supports the development of standardized, high-content platforms for investigating liver metabolism, toxicity, and therapeutic response in human disease research and drug discovery.</p>