BACKGROUND: Streptomyces rochei (ATCC 10739) has been reported for the production of a highly biologically active polyketide macrolide, i.e. borrelidin. The main objective of the present study was to develop an efficient fermentation, detection and quantification method for the production of borrelidin. RESULTS: Here, we report a rapid, sensitive and precise liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification and validation of borrelidin from S. rochei in the presence of other possible constituents in the fermentation broth. The method was validated as per guidelines proposed at the International Conference on Harmonization. CONCLUSIONS: A chromolith column and a multiple reaction monitoring LC-ESI-MS/MS method has been used for the first time for the separation and quantification of the borrelidin from the crude extract obtained from fermented broth of S. rochei. (C) 2014 Society of Chemical Industry