Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding siteof human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimentalconditions (HEPES buffer 50 mM, pH 7.4 and 25◦C) by means of isothermal titration calorimetry (ITC),fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique isbased on measurements of a different property of the biochemical system, and then the results allow acritical discussion about the suitability of each approach to estimate the drug-protein binding parameters.The strongest interaction step is properly evaluated by the three assayed approaches being the derivedbinding constants strongly consistent: from 4 × 104to 7 × 104for HSA and from 0.8 × 105to 1.2 × 105forBSA. Binding enthalpy variations also show consistent results: −5.4 and −5.6 Kcal/mol for HSA and −4.3and −3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction eventsfor both albumins are detected only by FA/CE.