Tetrodotoxins (TTXs) are one of the most potent marine neurotoxins known, and pufferfish are their main vector in nature. The toxicity of TTXs relies on the blockage of voltage-gated sodium channels (VGSCs) in neuronal or excitable cells, producing severe gastrointestinal and neurological symptoms that, in some cases, may be fatal. Until now, more than 30 TTX analogues have been described, but little is known about their role in poisoning. In this work, we propose the use of a high-throughput biosensing device for the detection of TTX and its analogues in pufferfish by assessing the electrophysiological activity of Neuro-2a cells exposed to fish extracts. The system showed an IC₅₀ value of 6.4 nM for TTX and, since detection was not affected by the presence of matrix components from different tissues of a TTX-free pufferfish specimen when analysed at 10 mg/mL, an LOD of 0.05 mg TTX equiv./kg was achieved (far below the Japanese regulatory limit of 2 mg TTX equiv./kg). The system was also used to evaluate the toxicity equivalency factors (TEFs) of five TTX analogues purified from the liver of a Lagocephalus sceleratus specimen fished near Crete (Greece). Then, naturally contaminated pufferfish samples were analysed using the single-cell biosensing device and compared with the LC-MS/MS quantifications after applying the TEFs, obtaining excellent correlations. Overall, the biosensing device presented in this work provides a rapid, highly sensitive and reliable toxicological method for the assessment of TTX and its analogues in seafood to protect consumers from TTX poisoning.