Isothermal titration calorimetry (ITC) is a well-established and convenient label-free technique for measuring biomolecular interactions in aqueous solutions. ITC enables accurate measurement of the affinities and thermodynamic parameters, such as changes in Gibbs energy, enthalpy, and entropy, which help dissect the binding mechanisms. ITC is commonly used to study protein–protein, protein–peptide, protein–DNA, and small-molecule inhibitor binding to target proteins. ITC can determine binding affinities ranging from nM to low-mM. This chapter provides a detailed protocol to investigate the binding of FOXO3 peptide to 14-3-3ε using ITC.