The production and certification of a series of azaspiracid (AZA) calibration solution reference materials is described. Azaspiracids were isolated from contaminated mussels, purified by preparative liquid chromatography and dried under vacuum to the anhydrous form. The purity was assessed by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The final concentration of each AZA in a CD(3)OH stock solution was determined by quantitative NMR spectroscopy. This solution was then diluted very accurately in degassed, high purity methanol to a concentration of 1.47 ± 0.08 μmol/L for CRM-AZA1, 1.52 ± 0.05 μmol/L for CRM-AZA2, and 1.37 ± 0.13 μmol/L for CRM-AZA3. Aliquots were dispensed into argon-filled glass ampoules, which were immediately flame-sealed. The calibration solutions are suitable for method development, method validation, calibration of liquid chromatography or mass spectrometry instrumentation and quality control of shellfish monitoring programs.