Mesenchymal Stem Cells (MSCs) could be a potential treatment for multiple diseases and injuries due to their ability to replenish many cell types in the human body. Translation of this requires cells to be maintained in their non-differentiated state, which remains a major challenge in the field. Optimal conditions for culturing these cells are now required; thus, in this study, rat MSCs cultured with three different supplemental serum concentrations were investigated using a proteomics approach. Bone marrow-derived rat MSCs were established in medium containing 10% serum. Cells were then starved prior to the application of media with different concentrations of serum (0%, 2% or 10%) and cultured for 24 hours. Cellular protein was collected and prepared for qualitative and quantitative (SWATH) mass spectrometry using standard techniques. Multivariate analysis (PCA, PLS-DA and oPLS-DA) revealed biochemical differences across treatments. Gene ontology (GO) enrichment analysis was used to determine the biological processes of cells in response to treatments. A protein library containing 803 proteins was generated, 58.03% of which were observed in all three treatments, while 8.47%, 8.47% and 1.49% were unique to 0%, 2% and 10% serum, respectively. Fewer proteins were detected in the 10% serum group compared to lower concentrations possibly due to high abundant protein induced ion suppression. Based on the biochemical data, there was a more pronounced similarity between the serum free and 2% treatments compared to the 10% treatment. However, it is clear that unique biochemical features exists within all three treatments and these features may provide the insight needed to monitor and maintain MSCs in long term culture.