A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated to identify and quantify trace levels of cyanotoxins or microcystins (MC) in water, bivalves and fish tissue with enhanced sensitivity and specificity. The method enables confirmation and quantification of six MCs (MC-LA, LF, LR, LW, RR and YR) with a single chromatographic run. The applied chromatography also allows determination of certain MC metabolites (Desmethyl-LR and -RR). By using LC-ESI-MS/MS in multiple reaction monitoring (MRM) mode, the limit of detection and quantitation for the microcystins studied, were determined to be between 0.2 and 1 pg on column (5:1 S/N ratio). These values are below the 2 pg detection limits found in the available literature.