Obtaining a detailed description of how active site flap motion affects substrate or ligand binding will advance structure-based drug design (SBDD) efforts on systems including the kinases, HSP90, HIV protease, ureases, etc. Through this understanding, we will be able to design better inhibitors and better proteins that have desired functions. Herein we address this issue by generating the relevant configurational states of a protein flap on the molecular energy landscape using an approach we call MTFlex-b and then following this with a procedure to estimate the free energy associated with the motion of the flap region. To illustrate our overall workflow, we explored the free energy changes in the streptavidin/biotin system upon introducing conformational flexibility in loop(3-4) in the biotin unbound (apo) and bound (holo) state. The free energy surfaces were created using the Movable Type free energy method, and for further validation, we compared them to potential of mean force (PMF) generated free energy surfaces using MD simulations employing the FF99SBILDN and FF14SB force fields. We also estimated the free energy thermodynamic cycle using an ensemble of dosed-like and open-like end states for the ligand unbound and bound states and estimated the binding free energy to be approximately -162 kcal/mol (experimental -18.3 kcal/mol). The good agreement between MTFlex-b in combination with the MT method with experiment and MD simulations supports the effectiveness of our strategy in obtaining unique insights into the motions in proteins that can then be used in a range of biological and biomedical applications.