The global need for accurate and sensitive quantitation of microcystins (MCs) persists as incidents of cyanobacterial harmful algal blooms continue to rise and recent research reveals an underestimation of the human health implications of these toxins. An optimal approach for their accurate quantitation relies on the availability of stable isotope-labeled MC standards for use in stable isotope dilution analysis (SIDA) strategies involving liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to the dearth of isotopically labeled MCs, ten different 15N-enriched MCs were biosynthesized from producing cultures and fully characterized. This involved the comparative MS/MS fragmentation of natural abundance or unlabeled metabolites with their 15N-labeled congeners for improved confidence in product ion annotation. These results revealed a series of incorrect annotations described previously in the literature. In this manuscript, the biosynthesis of labeled microcystin is detailed, and their complete analytical characterization for prospective use in targeted SIDA applications, such as routine water testing is described.