2,2,2-Trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) are known to be secondary structure inducers of proteins. In order to determine the efficacy of TFE and HFIP in affecting the conformation of proteins when taken together, as compared to individually, we have studied the thermodynamics of unfolding of bovine serum albumin (BSA) in the presence of these alcohols along with the conformational characterization of the protein. A comparison of change in thermal transition temperature of the protein in the absence and presence of these alcohols in combination and individually shows that the (TFE+ HFIP) mixture is a stronger stabilizer of BSA up to a certain molality as compared to addition of their individual effects. The thermodynamics of effects of these alcohols on dithiotheitol reduced BSA were also studied. The enthalpies of interaction of TFE with HFIP in aqueous solution were measured by using isothermal titration calorimetry. The endothermic molar enthalpy of interaction of TFE with HFIP suggests that these alcohols do not strongly associate with each other through polar interactions. This is a possible explanation for their stronger effect on protein stability and conformation in combination rather than individually. The circular dichroism and fluorescence spectroscopic results provide evidence for the enhancement of the secondary structure of the protein by TFE and HFIP along with internalization of tryptophan residues in more hydrophobic environment. (C) 2018 Elsevier Ltd.