A method for the determination of noboviocin in milk was presented by high performance liquid chromatography(HPLC). The novobiocin in the spiked sample was extracted with methanol and evaporated under vacuum. After evaporating, the residue was mixed with distilled water for 2$mell$, filtrates with 0.45$mu extrm{m}$ acrodisc was injected into HPLC. The results obtained were as follows ; 1. The calibration curve of novobiocin was showed constantly linear(r 0.999) in the range of 100~500ng/$mell$. 2. The mean recovery rate of novobiocin from the spiked milk sample were 88~98%. 3. The coefficients of variation were 2.6~5.8% 4. The lowest detectable limit of novobiocin was 25ppb.