Inhibitor-induced conformational ensemble shifts in a multidrug resistant HIV-1 protease variant, MDR769, are characterized by site-directed spin labeling double electron–electron resonance spectroscopy. For MDR769 compared to the native enzyme, changes in inhibitor IC50 values are related to a parameter defined as |ΔC|, which is the relative change in the inhibitor-induced shift to the closed state. Specifically, a linear correlation is found between |ΔC| and the magnitude of the change in IC50, provided that inhibitor binding is not too weak. Moreover, inhibitors that exhibit MDR769 resistance no longer induce a strong shift to a closed conformational ensemble as seen previously in the native enzyme.