Harmful algal blooms (HABs) and their toxins are a significant and continuing threat to aquatic life in freshwater, estuarine, and coastal water ecosystems. Scientific understanding of the impacts of HABs on aquatic ecosystems has been hampered, in part, by limitations in the methodologies to measure cyanotoxins in complex matrices. This literature review discusses the methodologies currently used to measure the most commonly found freshwater cyanotoxins and prymnesins in various matrices and to assess their advantages and limitations. Identifying and quantifying cyanotoxins in surface waters, fish tissue, organs, and other matrices are crucial for risk assessment and for ensuring quality of food and water for consumption and recreational uses. This paper also summarizes currently available tissue extraction, preparation, and detection methods mentioned in previous studies that have quantified toxins in complex matrices. The structural diversity and complexity of many cyanobacterial and algal metabolites further impede accurate quantitation and structural confirmation for various cyanotoxins. Liquid chromatography–triple quadrupole mass spectrometer (LC–MS/MS) to enhance the sensitivity and selectivity of toxin analysis has become an essential tool for cyanotoxin detection and can potentially be used for the concurrent analysis of multiple toxins.