Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum, that have been shown to cause diarrhetic shellfish poisoning when accumulated in bivalve molluscs. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the potential formation of AZA methyl-esters as artefacts in the extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures, using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl-ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl-esters. Hence centrifugation is recommended for recovery of cells. The type of extraction solvent (MeOH, Acetone, ACN) did not significantly affect the yield of AZAs so long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl-ester artefacts. AZA1 recovery over two successive extractions was 100% at 95% confidence level for acetone and MeOH. In standard addition experiments no significant matrix effects were observed in extracts of A. spinosum or A. obesum up to a sample intake of 4.5*109 µm3. Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues, led to the description of two new AZAs and the correction of the chemical structures of AZA29-32.