Wharton’s Jelly mesenchymal stem cells (WJ-MSC) are stem cells from the umbilical cord, known for their regenerative properties. They offer advantages like unlimited availability, non-invasive and low-cost isolation, and minimal ethical concerns. WJ-MSCs are promising for allogeneic cell therapy, with clinical trials showing their safety and efficacy across various diseases. They are also considered non-tumorigenic. A critical step for clinical use is selecting the optimal isolation method. This study compares three techniques: two explant methods (with and without scraping) and one enzymatic method. Additionally, the effect of adding fibroblast growth factor 2 (FGF2) to enhance cell expansion is examined. While several studies have addressed differences in isolation methods, the impact on the protein profile of WJ-MSCs is less explored. Therefore, we aim to explore the proteomic profiles of WJ-MSCs isolated by different methods to better understand their mechanisms and functionalities