The increased production of ethanol in the US has resulted in large amounts of distillers grains (DG) which is an excellent feed supplement for livestock. However, the use of antimicrobials during ethanol fermentation has led to a growing concern over the possibility of their residues remaining in DG. To enable the detection of antimicrobial residues, a robust LC-MS/MS method was developed that included 13 antibiotics of diverse chemistries, ampicillin, penicillin G, tetracycline, oxytetracycline, chlortetracycline, bacitracin A, virginiamycin M1, chloramphenicol, erythromycin A, clarithromycin, tylosin A, monensin A and streptomycin. The residues were extracted with an aqueous solution of EDTA and trichloroacetic acid followed by methanol. The combined extract was subjected to a two-track cleanup and concentration on either hydrophilic polymeric or weak cation exchange solid phase extraction cartridges. The extracts are analyzed by LC/ion trap tandem mass spectrometry. The method was validated in dry DG matrix. Absolute recoveries of the analytes ranged from 50 to 100%. Accuracy ranged from 89 to 111% based on calibration by processed standards. The limits of detection and relative standard deviation are satisfactory to support future surveillance studies. The method was subsequently tested in three different end-products of DG: distillers dry grains, distillers wet grains and distillers grains solubles.