Regenerative medicine brings promising applications for mesenchymal stem cells, such as dental pulp stem cells (DPSCs). Confocal Raman microscopy, a noninvasive technique, is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800 to 3000 cm⁻¹ region (C-H stretching) and the 960 cm⁻¹ peak (ν₁ PO₄³⁻) were collected (to image cells and phosphate, respectively), and the ratio of two peaks 1660 over 1690 cm⁻¹ (amide I bands) to measure the collagen cross-linking has been calculated. Raman spectra of DPSCs after 21 days differentiation reveal several phosphate peaks: ν₁ (first stretching mode) at 960 cm⁻¹, ν₂ at 430 cm⁻¹, and ν₄ at 585 cm⁻¹ and collagen cross-linking can also be calculated. Confocal Raman microscopy enables monitoring osteogenic differentiation in vitro and can be a credible tool for clinical stem cell based research.