This study determined the appropriate biochemical assay for measuring plasma tartrate-resistant acid phosphatase isoform 5b (TRAP5b) activity; this information is important to clarify the relationship between plasma TRAP5b and known biochemical bone markers in cattle. When plasma TRAP5b was measured using fluorometric and spectrophotometric methods, hemolysis products in plasma did not affect the former method. In plasma from healthy cattle, there was a good correlation (r=0.66) between the 2 methods. In age-related profiles, plasma TRAP5b (r=-0.53), hydroxyproline (HYP, r=-0.56) and bone-specific alkaline phosphatase (BALP, r=-0.44) showed significant negative correlations with age; these three parameters decreased until 4 or 5 years of age and then remained constant. There were significant correlations between TRAP5b and HYP (r=0.83) or BALP (r=0.83). Our results show that the fluorometric assay can be performed with a high degree of precision and reproducibility without interference from hemolysis, and that the age-related changes in plasma TRAP5b, HYP, and BALP constitute additional background values for clinical guidance in bovine medicine.