Novobiocin is a coumarin-containing antibiotic, and has a longer half-life in various animals than other veterinary medicines. A simple and rapid high-performance liquid chromatography assay for the determination of residual novobiocin levels in chicken, beef and milk has been developed and validated. The separation condition for HPLC/UVD was optimized by a MG II $C_{18}$ (4.6 mm $ID{ imes}250$ mm, 5 ${mu}m$) column with 0.1% formic acid in $H_2O$/0.1% formic acid in Acetonitrile (40/60, v/v) as the mobile phase at a flow rate of 1.0 mL/min and the detection wavelength was set at 340 nm. Residues were extracted from tissue by blending with methanol. After liquid-liquid partitioning, lipid materials were removed with n-hexane and purification as Silica (1 g, 6 mL) cartridge with 10 mL acetone/dichloromethane (10/90, v/v). Limit of quantification and linearity performed by the analytical method were 0.02 mg/kg and 0.999 ($r^2$), and the recovery range was $88.8{pm}5.6-100.3{pm}4.4$, $88.8{pm}7.2-97.0{pm}3.2$ and $88.1{pm}4.3-92.8{pm}3.6%$. It is expected that this analytical method with regards to novobiocin in chicken, beef and milk could be applied as an official method to administer food safety on veterinary medicines.