<jats:sec><jats:title>RATIONALE</jats:title><jats:p>A rapid and precise analytical method for the investigation of natural products is required for pathway monitoring of the biosynthesis of secondary metabolites. Phenalinolactones, used in antibiotic research, are produced by <jats:italic>Streptomyces</jats:italic> sp. Tü6071. For the analysis of those compounds, prior to mass spectrometric analysis, an efficient separation technique is required.</jats:sec><jats:sec><jats:title>METHODS</jats:title><jats:p>For the identification of phenalinolactones from liquid cultures of <jats:italic>Streptomyces</jats:italic> sp. Tü6071, a new method comprising the combination of solid‐phase extraction (SPE) prior to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) was established. MS/MS product ion scans were applied for phenalinolactone detection and structure elucidation, performed in negative mode and optimized for sensitivity and specificity. For the discovery of new intermediates, a MS/MS precursor ion scan was applied.</jats:sec><jats:sec><jats:title>RESULTS</jats:title><jats:p>Analysis of the extracts revealed that the Oasis® MAX cartridge, containing a quaternary amine functionality, is the most efficient SPE material for purification of phenalinolactones, since it allowed sufficient enrichment and detection of intermediates from the biosynthetic pathway by LC/ESI‐MS/MS. Using the precursor ion scan technique, two new secondary metabolites, PL IM1 with <jats:italic>m/z</jats:italic> 672.6 and PL IM2 with <jats:italic>m/z</jats:italic> 433.3, have been detected. The structures of the new intermediates are postulated and arranged into the biosynthetic pathway of phenalinolactones.</jats:sec><jats:sec><jats:title>CONCLUSIONS</jats:title><jats:p>A precise analytical method was established for the identification of phenalinolactones by combining purification from <jats:italic>Streptomyces</jats:italic> using SPE prior to LC/ESI‐MS/MS. By optimising LC/ESI‐MS/MS settings, this method has been successfully applied for pathway monitoring of secondary metabolites. Application of a precursor ion scan allowed for the identification of unknown intermediates in biosynthetic pathways. Copyright © 2014 John Wiley & Sons, Ltd.</jats:sec>