Phenotypic drug discovery enables the evaluation of anticancer drug responses without requiring prior knowledge of molecular targets. Tumor spheroids more accurately recapitulate in vivo tumor architecture compared to conventional monolayer cultures but currently lack standardized drug evaluation criteria. In this study, we fabricated uniform and drug evaluation–optimized tumor spheroids using triple-positive (ER+, PR+, HER2 +) invasive ductal carcinoma cells. Anticancer drugs used in breast cancer were administered at clinically relevant plasma concentrations. We propose drug response evaluation parameters including spheroid size change, morphological features (surface cell size and convexity), and optical transmittance monitored during drug treatment and throughout a defined post exposure observation period. These optical image–based analytical features showed consistent correlations with the respective mechanisms of molecular action and the associated drug response. This study establishes a phenotype-based evaluation system for breast cancer chemotherapy using tumor spheroids, offering a reference model for drug evaluation. Moreover, it proposes quantifiable evaluation parameters for drug screening and provides reference data for assessing investigational compounds, thereby enhancing the utility of spheroids in phenotypic anticancer drug discovery.