Azaspiracid (AZA-40) produced by the dinoflagellate Azadinium poporum is isobaric with the regulated analogue AZA-1 and has been detected across a wide geographic range. In this study, an A. poporum strain originally isolated from the South China Sea was mass cultures, and AZA-40 was subsequently purified and structurally characterized by NMR spectroscopy and high-resolution mass spectrometry. NMR analysis confirmed the high structural similarity between AZA-40 and AZA-1, with AZA-40 containing an additional methyl group at C-8 and lacking the methyl group at C-39. AZA-40 was approximately eight times more potent than regulated AZA-1 in inducing the oxidative stress response in the AREc32 assay and four times more cytotoxic in vitro, as measured by cell confluency. Activation of the oxidative stress response is indicative of exposure to highly reactive, and in some cases genotoxic, chemicals and is a useful proxy for adaptive stress responses that may occur in vivo. The high in vitro toxicity of AZA-40, in conjunction with the wide geographic distribution of the AZA-40-producing dinoflagellate A. poporum, suggests that this compound should be should be closely monitored in plankton and shellfish to assess its potential risk to food safety. The monitoring of novel AZAs by LC-MS typically requires reference standards for each compound. However, owing to the high structural similarity between AZA-40 and AZA-1, quantification of AZA-40 by LC-MS/MS yielded good results when AZA-1 was used as an external reference standard.