Mesenchymal stem cells (MSCs) have garnered attention as promising candidates for treating degenerative diseases; however, concerns persist regarding their stability and viability in cell therapy applications. In response, this study endeavors to augment MSC viability and efficacy by subjecting them to dimethyl fumarate (DMF) preconditioning, a process evaluated through phosphoproteomic analysis. To delve into the effects of DMF preconditioning, the researchers conducted protein extraction and digestion procedures on both untreated and DMF-treated MSCs. Utilizing the s-trap method alongside trypsin/lysin enzymes ensured comprehensive processing of the samples. Following this, phosphorylated peptides were enriched using TiO2 and identified via nanoLC-MS/MS technology. The resulting data underwent thorough analysis utilizing Maxquant and Perseus software tools. Remarkably, the analysis revealed a notable increase in the expression levels of 559 phosphorylated peptides among the 837 commonly detected in DMF-treated MSCs compared to their untreated counterparts. Network analysis conducted through Reactome shed light on RNA Binding Motif Protein 39 (RBM39) as a potentially pivotal mediator of cellular responses, hinting at its involvement in crucial RNA processing events and gene expression regulation mechanisms. Furthermore, the efficacy of TiO2 enrichment in boosting the fraction of phosphorylated peptides by approximately 50% underscores its robustness as a technique. In conclusion, this study serves as a testament to the significant impact of DMF treatment on the phosphoproteomic landscape of MSCs. The proposed avenue for further exploration of identified pathways via high-throughput phosphoproteomics promises a deeper and more comprehensive understanding of the underlying molecular mechanisms driving the observed effects.