Deep eutectic solvents (DESs), and particularly their mixtures with water, have been postulated as progressive, sustainable biocatalytic media. Currently, however, knowledge of biomolecular stability within DES media, such as protein folding reversibility, remains quite limited. In this Letter, we present the findings of a study of bovine ribonuclease A (RNase A) unfolding/refolding within aqueous media containing 5–35 wt % of illustrative DESs comprising 1:2 molar ratios of choline chloride plus urea (so-called reline), ethylene glycol (ethaline), or glycerol (glyceline). Using differential scanning calorimetry, iterative thermal cycling of RNase A in the presence of reline is shown to result in rapid and complete loss in folding reversibility, tentatively linked to ammonia evolution, whereas the addition of glyceline actually improves the RNase A thermodynamic stability beyond the native, purely aqueous milieu.