This study describes determination of chloramphenicol (CAP) in honey, milk, and egg by LC-electrospray ionization (ESI)-MS. CAP was extracted with ethyl acetate from honey, and with acetonitrile from milk and egg. d5-CAP was used as the internal standard. Separation was achieved on a ZORBAX Eclipse C18 2.1 id, 150 mm, 2.1 pm column. The mass spectrometer was equipped with an ESI interface in negative mode. Mass determination of CAP and d5-CAP was carried out in selected ion monitoring mode by scanning m/z 321 for CAP and m/z 326 for d5-CAP as quantification ions, and m/z 323 as the confirmation ion. The validation of the analytical method was performed by carrying out linearity, LOD, LOQ, accuracy, precision, and recovery. Calibration curve was linear between 0.1 and 20.0 ng/g with correlation coefficient >0.998. Accuracy of the method for the honey sample was 95%, and for the milk powder sample 106%. Precision of the method, expressed as CV%, varied between 1.60 and 4.37 for intraday and 2.00 to 5.67 for interday. Quantification limits of method were 0.05, 0.08, and 0.09 ng/g for egg, milk, and honey, respectively. Recovery of the method ranged between 86 and 103% for all three matrixes and two different concentrations. Finally, the method was checked by participating proficiency tests from the Food Analysis Performance and Assessment Scheme 0281 and 0298 honey samples. Z scores were -1.0 and 0.4, respectively.