Enrofloxacin is one of antibiotics as a class of fluoroquinolones and commonly used in intensive poultry farming to treat chronic respiratory disease, colibacillosis, and fowl cholera. However, the use of fluoroquinolones and other antibiotics for food-producing animals is now regulated and many countries have established maximum residue limits (MRLs) in their food products to safeguard human health from the risks of multi-resistant and aggressive bacteria. Accordingly, a validated analytical method as well as certified reference materials (CRM) for the evaluation of the method are required. Recently, CRM (KRISS CRM 108-03-003) have been developed by Korea Research Institute of Standards and Science. They have presented the concentration of enrofloxacin for CRM through the measurements of ten bottles in 10 g portions were taken randomly from the bulk meats ready-made in powders. The concentrations of the measurement results of ten bottles were calculated by one point calibration with isotope-ratio matching. The adoption of analytical method can be officially approved on the basis of a method’s performance in the inter-laboratory collaborative studies and the comparison of these results is an external way of assuring quality control among laboratories concurrently. Thus, three laboratories were involved in an inter-laboratory collaborative study to test for the method evaluation using the CRM of enrofloxacin in chicken meat. The common method, a liquid–liquid extraction combined with clean-up procedure based on solid-phase extraction (SPE) followed by LC-MS/MS has been performed by three individual laboratories. Linearity, limit of detection (LOD) and limit of quantification (LOQ) were assessed and the laboratory variance of results for CRM was compared. The inter-laboratory comparison results showed approximately 95 % of z-scores have fallen within two standard deviations which is commonly designated as an acceptable range.