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Abstract In search of effective therapeutics for breast cancers, establishing physiologically relevant in vitro models is of great benefit to facilitate the clinical translation. Despite extensive progresses, it remains to develop the tumor models maximally recapturing the key pathophysiological attributes of their native counterparts. Therefore, the current study aimed to develop a microsphere‐enabled modular approach toward the formation of in vitro breast tumor models with the capability of incorporating various selected cells while retaining spatial organization. Poly (lactic‐co‐glycolic acid) microspheres (150‐200 mm) with tailorable pore size and surface topography are fabricated and used as carriers to respectively lade with breast tumor‐associated cells. Culture of cell‐laden microspheres assembled within a customized microfluidic chamber allowed to form 3D tumor models with spatially controlled cell distribution. The introduction of endothelial cell‐laden microspheres into cancer‐cell laden microspheres at different ratios would induce angiogenesis within the culture to yield vascularized tumor. Evaluation of anticancer drugs such as doxorubicin and Cediranib on the tumor models do demonstrate corresponding physiological responses. Clearly, with the ability to modulate microsphere morphology, cell composition and spatial distribution, microsphere‐enabled 3D tumor tissue formation offers a high flexibility to satisfy the needs for pathophysiological study, anticancer drug screening or design of personalized treatment.