A sensitive and reliable method using gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS) was developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) at trace levels in muscle and liver. Before extraction with ethyl acetate, CAP-d(5) was added to tissue samples as internal standard. The organic extracts were frozen to remove lipid and further purified by liquid-liquid extraction (LLE) with hexane and solid-phase extraction (SPE) using Oasis HLB cartridges. The target compounds were derivatized with BSTFA+1% TMCS prior to GC-NCI/MS determination in selected ion monitoring mode (SIM). The recovery values ranged from 78.5 to 105.5%, with relative standard deviations (RSD) <17%. The limits of detections (LODs) of 0.1 microg/kg for CAP and 0.5 microg/kg for TAP, FF, and FFA were obtain. Incurred sample and samples from local market were successfully analyzed using this method.