Purpose <p>Stability of messenger RNA (mRNA) is a critical factor in the development and storage of mRNA-based vaccines. In this study, we systematically examine the effects of addition of various osmolytes (and their combinations) on mRNA integrity during long-term storage and under thermal stress.</p> Methods <p>Eleven osmolytes, belonging to subclasses of amino acids and derivatives (ectoine, methionine, L-cysteine, valine), polyols and sugars (sorbitol, trehalose and mannose), nucleosides (uridine), and vitamins and cofactors (nicotinamide and citric acid) have been examined by themselves as well in binary and ternary combinations. Their protective capacity has been evaluated. A concentration curve was developed where we assessed 0–30% weight/volume of the osmolytes in the mRNA vaccine formulations and analyzed the thermostability for 5–14&#xa0;days in 40°C and 2.5&#xa0;months at 4°C.</p> Results <p>An optimized formulation of 80:20 taurine to uridine showed robust mRNA protection across 15–30% w/v concentrations (densitometric indices 0.833–0.875, <i>p</i> &lt; 0.001) with exceptional stability at 1% w/v (0.966 ± 0.061) after 5&#xa0;days at 40°C, while the ternary taurine-uridine-sorbitol system achieved peak performance at 5% w/v (0.917 ± 0.036). This established a concentration-dependent design principle for thermostable mRNA formulations with &gt; tenfold improvement over untreated controls under accelerated thermal stress conditions.</p> Conclusions <p>Our results revealed that the effectiveness of each component depended not only on its identity but also on the composition of the formulation. Ternary mixtures did not always outperform binary systems. Our work demonstrates that osmolytes offer significant potential towards improving the shelf life and accessibility of mRNA vaccines.</p> Graphical Abstract <p>Osmolyte-Mediated Thermal Stabilization of mRNA Vaccines</p> <p>This schematic illustrates the protective mechanism of osmolyte formulations for mRNA vaccine stabilization under thermal stress. Native mRNA vaccines (left) are combined with systematically selected osmolyte formulations to assess enhancement in stability when exposed to thermal stress. Osmolyte-protected mRNA is seen to maintain its structural integrity with an intact backbone (top right), while unprotected mRNA suffers significant degradation and fragmentation (bottom right). The color-coded matrix represents the diverse osmolyte classes and combinations evaluated in this study, highlighting the rational design approach used to identify optimal protective formulations for enhanced mRNA vaccine thermostability.</p> <p></p>