A sensitive and reliable method using liquid chromatography–negative electrospray ionization mass spectrometry was developed for the simultaneous determination of chloramphenicol, florfenicol, and thiamphenicol at trace levels in animal feed. The analytes were extracted from grinded feed with ethyl acetate. Further the ethyl acetate was evaporated, residue resuspended in Milli-Q water, defatted with n-hexane, and solid phase extracted using BondELUT C18 cartridges. Separation was carried out on a C6 phenyl column with a mobile phase consisting of 0.1% formic acid in Milli-Q water and acetonitrile. The detector response was linear over the tested concentration range from 100 to 1000 μg kg−1. The recovery values for all analytes in feed were higher than 79% with RSD for repeatability and reproducibility in the ranges of 4.5–10.9% and 8.4–13.5%, respectively. CCα and CCβ varied between 76.8 and 86.1 μg kg−1, and between 111.3 and 159.9 μg kg−1, respectively. The results showed that this method is effective for the quantification of phenicols in non-target feed.