Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing MSC self-renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub-cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow-derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two-Dimensional Liquid Chromatography/Matrix-Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self-renewal and differentiation.