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Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 μg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 μg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.