Prokaryotic organisms such as cyanobacteria are a large, diversified species that can both photosynthesis and breathe through heterotrophic routes. A significant problem with water quality that has an international impact on surface water management is cyanobacterial blooms. There is potential for a wide range of hazardous secondary metabolites to be produced by numerous aquatic cyanobacterial blooms. One example of a neurotoxic made by cyanobacteria is anatoxin. It has already been noted that some species of the genera Anabaena and Oscillatoria are capable of producing anatoxin. Anatoxin is a powerful cyanotoxin-like alkaloid that blocks neuromuscular transmission by depolarizing the post-synaptic membrane potential. In this protocol, we practiced about extraction and quantification of anatoxin from aquatic sample. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023.