The aim of this work was to develop a method to purify large amounts of spirolide toxins from cultures of Alexandrium ostenfeldii. The dinoflagellates grew in batches under controlled conditions of salinity, light and temperature. Analysis of the cultures demonstrated the existence of neurotoxins associated with paralytic shellfish poisoning toxins and two spirolides, 13-desmethyl spirolide C and 13,19-didesmethyl spirolide C. The protocol designed presents several stages of extraction, separation between spirolides and paralytic shellfish poisoning toxins, and cleanup in solid-phase extraction. Finally, the purification of spirolides was conducted by a preparative high-performance liquid chromatography system coupled to a mass spectrometer detector. The purity and the amount of both toxins in each step was monitored by analytical liquid chromatographic-mass spectrometry. Large amounts of 13-desMeC, 97% pure, and 13,19-didesMeC, 99% pure, were obtained. A novel and efficient method to separate and purify spirolide toxins from large amounts of phytoplankton is provided. The protocol proposed shows, for the first time, a complete and detailed methodology to separate and purify spirolide toxins with high purity, recovery, repeatability and stability.