A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of tetracycline (TC) residue in biological fluids. This method allows detection of residual TC in biological fluids by using singleionmonitoring (SIM), confirmation by a full scan electron impact (EI) mass spectrum is possible if residual level in a sample is >1 µg/mL. TC is extracted with chloroformfroma sample and cleaned up by n-hexane washing followed by partition between chloroform and phosphate buffer solution. The cleaned up extract is acetylated in acetic anhydride-pyridinemixture (1: 2) at room temperature. The reaction mixture is injected into the GC-MS apparatus, and the detection has been conducted using SIM at m/z 149. The detection limit is 0.01µg/mL. This method could be adopted in the forensic laboratories as well as in the toxicological laboratories.