Numerous efforts have been made to establish three-dimensional (3D) cell cultures that mimic the structure, cell composition, and functions of actual tissues and organs in vitro; however, owing to its intrinsic complexity, precise characterization of 3D differentiation remains challenging and often results in high variations in the resulting differentiated spheroids. This study reports a 3D Raman mapping-based analytical method that helps us to identify the crucial factors responsible for inducing variability in differentiated stem cell spheroids. Human dental pulp stem cell spheroids were generated at various cell densities using the hanging drop (HD) and molded parafilm-based (MP) methods and were then further subjected to odontogenic differentiation. Thereafter, the 3D cellular differentiation in these spheroids was analyzed based on three different Raman peaks, namely, 960, 1156/1528, and 2935 cm-1, which correspond to hydroxyapatite (HA, odontogenic differentiation marker), β-carotene (precursor of HA), and proteins/cellular components (cell reference). By correlating such cell differentiation-related peaks and water/medium peaks (3400 cm-1), we discovered that the diffusion of the medium containing various nutrients and differentiation factors was crucial in determining the variations in 3D differentiation of stem cell spheroids. Odontogenic differentiation was majorly induced at the outer shell of HD spheroids (up to ∼20 μm), whereas odontogenic differentiation was markedly induced in MP spheroids (up to 40-50 μm). Considering the challenges associated with high variations in spheroid and organoid differentiation, we conclude that the proposed Raman-based 3D analysis plays a pivotal role in stem cell-based regenerative therapy and drug screening.