Novobiocin is used for the treatment of mastitis in dairy cattle. In 1982, the tolerance was set at 0.1 ppm in milk from dairy animals. A laboratory trial has been completed for a liquid chromatographic procedure that can quantitate novobiocin residues in bovine milk at tolerance level. In this procedure the milk is diluted with buffer, the proteins are precipitated with methanol, and the solution is filtered. Novobiocin is determined after separation of milk components using reversed-phase chromatography with UV detection at 340 nm. The participating laboratories analyzed 2 concentrations of biologically incurred residues as well as control milk and control milk fortified at 0.05, 0.1, and 0.2 ppm. Recoveries of novobiocin reported by the participating laboratories were 89 to 99% at 0.05 ppm; 93 to 101% at 0.1 ppm; and 89 to 100% at 0.2 ppm. Coefficients of variation (CVs) ranged from 2.0 to 6.2%. The average concentrations for the low levels of incurred novobiocin in milk samples were 0.073, 0.072, and 0.081 ppm, with intralaboratory CVs of 3.3, 7.2, and 2.4%, respectively. The samples with high levels of incurred novobiocin averaged 0.139, 0.121, and 0.144 ppm, with CVs of 6.2, 0.7, and 4.7%, respectively.