Bacitracin, colistin as well as polymyxin B are well-known antibiotics that for a long time have played a minor role in human medicine due to their toxicity. In contrast they were often used in veterinary medicine. With increasing development of drug resistance pathogenic bacteria the mentioned polypeptide antibiotics regain importance and an increased control of the maximum residue limits (MRLs) established by the European Commission in foods is required. Limits are set for the sum of bacitracin A, B and C and for the sum of colistin A and B, while polymyxin B is not regulated. In this work liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was applied to for the detection of bacitracin A and B, colistin A and B as well as polymyxin B1 and B2 in milk and animal tissue samples. Different protocols for sample preparation were evaluated, various chromatographic separation conditions tested, as well as mass spectrometric settings optimised. The developed analytical method is divided into the following stages; (1) sample preparation, based on acidic solvent extraction and followed by solid phase extraction (SPE) and purification using HR-X cartridges, (2) chromatographic separation in reversed-phase conditions based on a gradient elution program within a total run time of 15 min. and finally (3) electrospray ionization tandem mass spectrometric measurement under selective multiple reaction monitoring (MRM). Acquisition of triple charged precursor ions for all analytes was performed. The method was successfully validated by an in-house protocol according to the requirements of 2002/657/EC in these matrices. The presented method will serve as a basis for the quantitative analysis of the concerned polypeptides in foods in an analytical routine laboratory.