Serum albumins are ubiquitous proteins able to bind a variety of exogenous and endogenous ligands including hydrophobic pharmaceuticals. Most drugs bind to two very active binding regions located within sub-domains IIA and IIIA of the protein, also known as Sudlow's sites. The drug binding mode of serum albumin provides important pharmacological information and influences drug solubility, efficacy, biological distribution, and excretion. Here, the binding thermodynamics of Diclofenac and Naproxen, two non-steroidal anti-inflammatory drugs (NSAIDs) to bovine and human serum albumins (BSA and HSA, respectively) were studied by isothermal titration calorimetry (ITC), fluorescence spectroscopy and differential scanning calorimetry (DSC). The ITC data show that the binding affinity (K) of Diclofenac to BSA and HSA is on the order of 104 M-1 with a binding stoichiometry (n) of 2 drug molecules per protein. Naproxen binding to the two proteins exhibits a different profile with K and n values on the order of 10(6) M-1 and 0.75 for BSA, and 105 M-1 and 3 for HSA, respectively. The binding of the two drugs to HSA is found to be both enthalpically and entropically favored suggesting the formation of hydrogen bonds and van der Waals hydrophobic effects. Binding of the two drugs to BSA is only enthalpically favored with an unfavorable entropy term. Significant enthalpy-entropy compensation phenomena were reported for Diclofenac and Naproxen binding to BSA but not to HSA. Fluorescence quenching data between Diclofenac and the two proteins suggest static collisions and the formation of ground-state protein-drug complexes. The DSC data corroborate the ITC findings and show multiple sequential unfolding events and a strong drug stabilization effect at high drug to protein ratios. Overall, the calorimetric and spectroscopic data provided insights into the nature of these protein-drugs interactions and might offer useful information in future drug discovery studies. (C) 2016 Elsevier Ltd.