The atropodiastereomeric dimeric naphthylisoquinoline alkaloids, michellamines A (1a), B (1b) and C (1c), together with their monomers, korupensamines A (2a) and B (2b), were investigated using electrospray ionization tandem mass spectrometry coupled to liquid chromatography (LC-ESI-MS-MS). From the spectra obtained, characteristic product ions were chosen to monitor the chromatographic separation achieved on an RP-18 column. Under acidic conditions required for chromatographic analysis, the monomeric alkaloids 2a and 2b yielded protonated molecules [M + H]+, while the dimers, the michellamines, exhibited doubly protonated [M + 2H]2+ molecules. In addition, the coeluting alkaloids 1b and 2b were identified unambiguously be means of tandem mass spectrometry. Thus, together with the retention times of the alkaloids, the product ion spectra allowed us the identification of michellamines in the presence of their presumed biogenetic monomeric precursors. Application of the HPLC-MS-MS method successfully proved the enzymatic formation of michellamine C (1c) by in vitro dimerization of korupensamine B (2b).