A new analytical method for the determination of colistin in fermenter samples was developed followed by a study on the behavior of this substance during anaerobic fermentation. Analysis of colistin A and B was carried out by liquid chromatography-tandem mass spectrometry. Separation of the analytes was performed on a Security Guard column (4 x 3 mm). Fourteen fermentation tests in batch as well as in continuous reactors were carried out. After 44 days of anaerobic digestion of cattle manure, initially spiked with 500 mg/kg of colistin sulfate, a considerable decrease of the colistin concentration to less than 1 mg/kg could be observed. Furthermore, the daily production of biogas and methane was measured. A correlation between gas production and colistin concentration could not be determined. However, an increase of 10% of the cumulative methane production was observed in those fermenters spiked with an initial bolus of 500 mg/kg colistin. (C) 2014 Elsevier Ltd. All rights reserved.