Relaxin (RLX) is a peptide hormone belonging to the relaxin-like peptide family. Relaxin-2 (RLX-2), a heteromeric polypeptide consisting of an A-chain (24 amino acids) and a B-chain (29 amino acids) linked together by two inter-chain disulfide bonds, is the main circulating RLX hormone in human. Due to its ability to dilate blood vessels surrounding the smooth muscles via induction of nitric oxide resulting in the increase of blood and oxygen supplies to the muscles, it may enhance athletic performance and is therefore banned in horseracing, equestrian competitions, and human sports. In order to control the abuse of rhRLX-2, a definitive method is required to detect and confirm the presence of rhRLX-2 in biological samples. This paper describes, for the first time, the detection and confirmation of rhRLX-2 in equine plasma by liquid chromatography-high resolution mass spectrometry (LC-HRMS) after immunoaffinity extraction. rhRLX-2 could be detected at less than 0.1 ng/ml, and confirmed at less than 0.2 ng/ml in plasma samples. Copyright © 2012 John Wiley & Sons, Ltd.