Honey is a natural food widely consumed due to its high content in nutrients and bioactive substances. In order to prevent hive infections, xenobiotics such as pesticides and antibiotics are commonly used. Chloramphenicol (CAP) is a broad-spectrum antibiotic used to treat honeybee larvae diseases. However, CAP has toxic and nondose-dependent effects in sensitive subjects; for this reason, its use has been prohibited in food-producing animals, such as the honeybee. In this study, we proposed a rapid, simple, and cheap analytical method, based on salting-out assisted liquid-liquid extraction coupled with UHPLC MS/MS detection for the accurate determination of CAP in honey to be used in routine analyses. The parameters that influence the extraction efficiency have been optimized using an experimental design in order to maximize the recovery of the analyte by reducing the matrix effects. Therefore, the developed method was internally validated according to the 2002/657/EC Decision guidelines and applied to the analysis of 96 honey samples.