A simple method for the determination of chloramphenicol in 22 matrices was prepared based on the QuEChERS and HPLC-MS/MS combination. Following a hydrolysis step, the homogenized samples were extracted and partitioned after adding sodium chloride with acetonitrile. Chloramphenicol was analysed by HPLC-MS/MS in negative electrospray mode by monitoring the daughter ions m/z: 321→194 and 321→152. The limit of decision (CCα) was calculated at the range of 0.10 μg kg−1 to 0.15 μg kg−1 and detection capability (CCβ) from 0.12 μg kg−1 to 0.18 μg kg−1. Validation results showed that this method is suitable for the determination and confirmation of chloramphenicol in various matrices