γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system in mammals and there’s evidence that GABA can be used to relax horses before the race. In addition to its endogenous presence, it should be noted that GABA is also present in certain plants (ex. sprouts in general). It is therefore necessary to establish an International Threshold in order to detect any administration of GABA. For this purpose a very fast liquid chromatography-mass spectrometry (LC/MS) quantitative method (dilute and shoot) was developed for the determination of GABA in plasma. The method was fully validated according to EHSLC guidelines. Utilizing this methodology, a total of 1000 equine plasma samples collected pre and post-competition from horses (male, geldings, female) participating in equestrian events in Italy were analyzed. The data obtained are consistent with other studies conducted to quantify the average concentration of GABA in equine plasma and a threshold of 250 ng/ml corresponding to a risk factor of less than 1 in 11.000 in plasma is proposed.