Micelle-assisted “deintercalation” of intercalated drug/mutagen molecules from DNA is a well-established phenomenon; however, the driving energy cost for such a process is still not properly understood. In the present contribution, we have estimated the various energetic parameters for the SDS micelle-assisted deintercalation of a model DNA intercalator phenosafranine (PSF) using isothermal titration calorimetry (ITC) measurement. Both steady-state and picosecond-resolved fluorescence measurements provide strong evidence for the relocation of PSF molecules from the DNA interior to the micellar interface at an SDS concentration above cmc. The overall deintercalation process has been found to be enthalpy-wise forbidden (endothermic); however, it is strongly favored by a high positive entropy change, which can be correlated with the change in the associated hydration structure at the macromolecular interface.