Objective To assess the toxicity of the pufferfish Takifugu oblongus, from Chennai coast, Tamil Nadu, India and to detect the presence of Tetrodotoxin (TTX). Methods The toxicity was evaluated by mouse bioassay using Swiss Albino mice which were expressed in mouse units (MU). Gross anatomical features were observed which is followed by histopathology of the dead mice tissues to establish the toxicity. Instrumental analysis for the presence of tetrodotoxin was also performed through GC–MS and HPLC. Results The toxicity of ovary was the maximum with 163 MU/g and lowest toxicity was observed in skin with 75.88 MU/g. Histopathological analyses of the dead mice showed various cellular degenerations and inflammations. The amount of Tetrodotoxin detected through GC–MS and HPLC was more reliable and sensitive than the customary mouse bioassay as instrumental analyses were able to detect even nanograms of the toxin. Conclusions The present study evidently proved that Takifugu oblongus is highly toxic and consumption of the same can pose serious threat for health and possible lethality to humans. © 2017 Hainan Medical University