Developing three-dimensional (3D) co-culture models that emulate the complex architecture and cell distribution of salivary gland tissue is critical for studying its complex physiology and disease mechanisms. In this study, we established and optimized a 3D co-cultured microtissue model using murine NIH 3T3 fibroblasts and SCA-9 submandibular gland salivary epithelial cells to investigate multi-cellular interactions characteristic of salivary gland tissue. Non-adherent well plates showed superior performance compared with the conventional hanging drop method for their consistency in yielding spheroids of controlled size. The optimal cell seeding density was as low as 160 cells per well, consistently producing spheroids smaller than 200 µm, the critical size to prevent necrotic core development and maintain spheroid roundness. LIVE/DEAD staining confirmed high cell viability and the absence of central necrosis. Immunostaining with vimentin for NIH 3T3 fibroblasts and TAS2R4 for SCA-9 salivary epithelial cells demonstrated a mixed cell distribution within the spheroids. Furthermore, we tested the feasibility of using the salivary gland spheroids for irradiation studies by exposing them to increasing X-ray doses, revealing a dose-dependent increase in apoptosis. This self-assembled, Matrigel and scaffold-free, 3D co-culture model replicates critical features of the salivary gland’s native microenvironment, providing a foundational tool for future functional and pathological studies.