The limited predictive value of conventional two-dimensional (2D) cell culture models remains a major obstacle to successful anticancer drug development. Although three-dimensional (3D) multicellular tumor spheroids better reproduce key structural and functional features of solid tumors, standardized methodologies and biologically relevant analytical approaches for evaluating therapeutic responses throughout spheroid maturation remain insufficiently established. We developed a simple, low-cost, and highly reproducible protocol for generating homogeneous A549 human lung adenocarcinoma spheroids in agarose-coated 96-well plates and systematically characterized their structural and proliferative maturation over 20 days in vitro (DIV). Based on coordinated changes in architecture and proliferative activity, spheroids were classified as early-(4 DIV), intermediate-(8–12 DIV), and late-maturation stage (16–20 DIV). Cisplatin and metformin were used as chemotherapeutic agents with distinct mechanism of action to investigate how spheroid maturation and analytical endpoint selection influence therapeutic response. Growth inhibition (GI ₅₀ ) was evaluated using image-based morphometric analysis, MTT reduction assay, and intracellular ATP quantification, while Ki67 expression was used to assess proliferative activity. The workflow consistently generated a single homogeneous spheroid per well by 4 DIV, followed by progressive increases in size, structural complexity, tissue compartmentalization and a decline in proliferation. Cisplatin sensitivity progressively decreased during spheroid maturation, and morphometric- and MTT-derived GI ₅₀ values were inversely correlated with Ki67 expression, whereas ATP quantification underestimated maturation-dependent resistance profile. Importantly, 2D A549 cultures did not reproduce a corresponding relationship between proliferation and cisplatin sensitivity. Conversely, metabolic viability assay such as MTT better identify the spheroid sensitivity against metformin. Collectively, these findings demonstrate that spheroid maturation reshapes chemotherapeutic response through an integrated phenotype and that analytical endpoints should be interpreted according to their biological concordance with drug mechanism of action. Standardizing maturation stage together with mechanism-informed endpoint selection may improve the reproducibility and mechanistic interpretation of 3D preclinical cancer models, supporting their implementation within New Approach Methodologies (NAMs) and the principles of Replacement, Reduction and Refinement (3Rs) in anticancer drug development.