An assay was developed and proven accurate and precise for the quantification of canine serum alkaline phosphatase of bone origin (BAP). The assay uses wheat germ lectin (WGL) which selectively precipitates BAP but not liver alkaline phosphatase (LAP) in serum preincubated for 1 hour at 37°C before conducting the assay. Although a large percentage of corticosteroid‐induced alkaline phosphatase (CAP) is also precipitated by WGL, the activity of this isoenzyme can be determined by utilizing the automated levamisole inhibition assay and BAP determined by subtraction except in those cases in which CAP is very markedly increased. Use of these two assay techniques in combination allows the quantification of LAP, BAP, and CAP activity in canine serum.In sera from adult dogs of various ages, BAP activity represents a mean of 21.27 ± 11.4 U/L; however, there was a statistical decrease in BAP activity with age. This allowed the determination of 95% confidence interval for a reference range dependent on age of the dog. Bone AP activity in puppies drops dramatically within the first 3 months, reaching a magnitude of activity consistent with that of the adult dog by approximately 15 months. BAP was increased over adult reference range in five of five dogs tested with osteosarcoma. This assay will now allow conducting a clinical study of the diagnostic significance of bone AP activity in neoplastic and metabolic diseases of b