The goal of this study was to compare the growth and differentiation potential of rhesus monkey mesenchymal stem cells (rhMSCs) from different age groups (fetal, newborn, infant, juvenile), and to use confocal micro-Raman spectroscopy to assess the intrinsic biomolecular profiles of individual rhMSCs. Results indicated that fetal cells had significantly shorter population doubling times during the log growth phase (23.3 ± 1.3 h) and greater population doubling times (66.5 ± 6.5) when compared to other age groups (newborn 51.9 ± 2.3, infant 38.2 ± 3.1, juvenile 40.7 ± 4.1). Fetal rhMSCs also differentiated toward osteogenic and adipogenic lineages at a faster rate when compared to cells from older animals. The Raman spectral analysis showed greater DNA and lower protein concentration in fetal compared to juvenile rhMSCs, although the spectra from different age groups shared many similar features. Additionally, principal component analysis (PCA), which is used to discriminate between rhMSCs, supported prior findings that suggested that cultured rhMSCs consist of a heterogeneous cell population. Although the growth potential of rhMSCs from the younger age groups was confirmed, further studies will be necessary to fully explore the potential usefulness of Raman micro-spectroscopy to characterize stem and progenitor cells such as rhMSCs.