A label-free surface plasmon resonance biosensor method was applied to determine tetrodotoxin (TTX) in pufferfish matrixes using an antibody inhibition assay format. A prevalidation study was conducted to demonstrate the assay performance characteristics, such as selectivity, LOD, LOQ, repeatability, reproducibility, and accuracy. Three participating laboratories reported standard curves in buffer and pufferfish matrix. A set of blind samples with TTX spiked into buffer as well as in 10% pufferfish extract were analyzed. Additionally, three blind naturally contaminated samples were analyzed, and the results were compared to those obtained using a reference method (HPLC/electrospray ionization-selected reaction monitoring-MS). The developed method was demonstrated to be capable of detecting TTX in pufferfish matrix standard samples in a broad concentration range (2-9000 ng/mL) with an LOD of 1.5 ng/mL. Between-laboratory recovery values were in the range of 51-190% with a mean of 107%, and 64-180% with a mean of 103% for TTX-spiked samples in buffer and pufferfish matrix, respectively. Between-laboratory recoveries were in the satisfactory range of 101-119% for naturally contaminated samples. This robust, rapid, and noninvasive method may serve as an attractive alternative to established methods for detection of TTX in pufferfish extracts.