Recent research suggests that human umbilical cord derived mesenchymal stem cells (hUC‐MSCs) can be promising candidates for cell‐based therapy. Since large population and high viability are generally required, detecting viability transitions of these cells is crucial for their population expansion and quality control. Here, as a non‐invasive method, Raman micro‐spectroscopy is applied to examine hUC‐MSCs with different viability. Using peak fitting and statistic t‐test, the Raman peaks with obvious differences between the cells with high viability (>90%) and low viability (<20%) are extracted. It is found that the C=O out of plane bending in thymine at 744 cm–1, symmetric stretching of C–C in lipids at 877 cm–1 and CH deformation in proteins at 1342 cm–1 show the most significant changes (p <0.001). When the cell viability decreases, the intensities of the former two peaks are both about doubled while that of the latter peak reduces by about 30%. Based on these results, we propose that the viability of hUC‐MSCs can be characterized by these three peaks. And their intensity changes can be understood from the model of excessive reactive oxygen species interacting with the biomacromolecules. (© 2011 by Astro Ltd., Published exclusively by WILEY‐VCH Verlag GmbH & Co. KGaA) (© 2010 by Astro Ltd., Published exclusively by WILEY‐VCH Verlag GmbH & Co. KGaA)