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Long viewed as the ‘method of choice’ for characterizing thermodynamics and stoichiometry of molecular interactions, with high sensitivity, isothermal titration calorimetry (ITC) has been applied to many areas of pharmaceutical analysis. This review highlights ITC employment to measure binding thermodynamics and their use for pharmaceutical formulations and drug delivery system characterization particularly cyclodextrin–guest interactions, investigation of micellar-based systems, polyelectrolytes, nucleic acid interactions with multivalent cations and the optimization of DNA targeting and delivery. Furthermore, the potential of ITC for the characterization of different functionalities carried by nanoparticles as well as their interaction with living systems was outlined.