Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) are a valuable tool in stem cell research due to their high proliferation rate, multi-lineage differentiation potential, and immunotolerance properties. However, fibroblast impurity during WJ-MSCs isolation is unavoidable because of morphological similarities and shared surface markers. Here, a proteomic approach was employed to identify specific proteins deferentially expressed by WJ-MSCs in comparison to those by neonatal foreskin and adult skin fibroblasts (NFFs and ASFs, respectively). EphA2, SLC25A4, and SOD2 were predominantly expressed by WJ-MSCs, while CDH2 and Talin2 were specific to NFFs and ASFs, respectively. Here, EphA2 was established as a potential surface-specific marker to distinguish WJ-MSCs from fibroblasts and for prospective use to prepare pure primary cultures of WJ-MSCs for prospective clinical use. Additionally, CDH2 could be used for a negative-selection isolation/depletion method to remove neonatal fibroblasts contaminating preparations of WJ-MSCs.