A human in vitro resorption assay has been developed using osteoclastoma‐derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the α_vβ₃ integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human α_vβ₃‐mediated cell adhesion assay for the vitronectin receptor antagonists (r² = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H⁺‐ATPase inhibitors (r² = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r² = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long‐term in liquid nitrogen and upon thawing maintain their bone‐resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24–48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C‐telopeptides or N‐telopeptides, as measured by enzyme‐linked immunosorbent assay. Resorption can be measured reproducibly using a 48‐h incubation of osteoclasts on bone slices, or a 24‐h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.