Biomacromolecules are inherently dynamic, and their dynamics are interwoven into function. The fast collective vibrational dynamics in proteins occurs in the low picosecond timescale corresponding to frequencies of ∼5–50 cm−1. This sub-to-low THz frequency regime covers the low-amplitude collective breathing motions of a whole protein and vibrations of the constituent secondary structure elements, such as α-helices, β-sheets and loops. We have used inelastic neutron scattering experiments in combination with molecular dynamics simulations to demonstrate the vibrational dynamics softening of HIV-1 protease, a target of HIV/AIDS antivirals, upon binding of a tight clinical inhibitor darunavir. Changes in the vibrational density of states of matching structural elements in the two monomers of the homodimeric protein are not identical, indicating asymmetric effects of darunavir on the vibrational dynamics. Three of the 11 major secondary structure elements contribute over 40% to the overall changes in the vibrational density of states upon darunavir binding. Molecular dynamics simulations informed by experiments allowed us to estimate that the altered vibrational dynamics of the protease would contribute −3.6 kcal mol−1 at 300 K, or 25%, to the free energy of darunavir binding. As HIV-1 protease drug resistance remains a concern, our results open a new avenue to help establish a direct quantitative link between protein vibrational dynamics and drug resistance.