We present a rapid and simple method for detecting novobiocin in biologic samples using a methanol-based extraction of the tissue matrix and liquid chromatography with electrospray tandem mass spectrometry (LC-ESI-MS/MS) on positive mode. The sample, prepared using centrifugal ultrafiltration with 5.0% SDS, was directly injected into the LC-MS/MS. Chromatographic separation was performed on a TSK-GEL ODS 100 V column using 0.5% formic acid in water/methanol. The method was validated according to the Japanese Maximum Residue Limits recommendations. Detection was linear over a range of 5-100 ppb matrix solution (r>0.998). Novobiocin recovery values from chicken (0.05 ppm) and fish tissues (0.05 ppm), milk (0.08 ppm), and human serum (0.05 and 0.01 ppm) samples ranged from 71+/-1 to 95+/-2%.