Per-N-15-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography-mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in (NO3)-N-15-containing TS-15 medium. To change from the incorporation of N-14 to N-15 into all cell components, cells of Microcystis aeruginosa were precultured in (NaNO3)-N-15-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-N-15-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-N-15-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-N-15-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98-106%) was obtained, with the m/z of M+, [M+1](+), and [M+2](+) of both microcystins and the per-N-15-labeled microcystins as surrogates being completely separated. In conclusion, per-N-15-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography-mass spectrometry.