AbstractCompatible solutes constitute a diverse class of low‐molecular‐mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like α, α‐trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R‐2‐O‐α‐D‐mannopyranosyl‐glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high‐temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2‐O‐α‐D‐glucopyranosyl‐sn‐glycerol) and glucosylglycerate (R‐2‐O‐α‐D‐glucopyranosyl‐glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of α,α‐trehalose. Glucosylglycerate in concentrations of ≥0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. α,α‐Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.