Cyanobacterial harmful algal blooms (cHABs) are increasing in frequency and magnitude. cHABs release mixtures of bioactive compounds into freshwater that negatively impact human and ecosystem health. Factors that influence cyanobacterial growth and toxin production are broadly understood. However, cHABs are a prolific source of structurally diverse and strain-specific mixtures of toxins. Currently, the chemistry, environmental concentrations, and risks associated with most cyanobacterial metabolites are unknown. We utilize untargeted and semi-targeted mass-spectrometry based metabolomics to decipher the strain-specific profiles of non-ribosomal peptide metabolites (cyanopeptides) from five Microcystis strains. The established semi-targeted approach enables detection of all variants of a cyanopeptide group within environmental samples. Finally, growth medium composition effects on Microcystis metabolomes are assessed with the metabolomic techniques. Optimal growth conditions for cyanopeptide production are elucidated and provide means for large-scale cultivation of target strains for isolation and characterization of novel and common environmental contaminants for structural and toxicological assessment.