During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = vst + ((vi - vs)/kobs)(1 - exp(-kobst)). The initial rate vi represents the residual activity of the enzyme before inhibitor dissociation; vs is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of vi/vs. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC50 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production.