Highly potent opiates such as Fentanyl and its analogs have a large potential for abuse in horse racing due to their combined pain-relieving properties and their ability to induce Central Nervous System (CNS) stimulatory effects. Recently, a large number of highly potent fentanyl analogs have been synthesized leading to concerns about the ability to detect these compounds in routine analysis. A standard post-race drug testing panel does not currently include many of these novel fentanyl analogs, therefore these may be undetectable in routine testing. Additionally, these compounds are highly potent and their presence in urine and blood samples may evade detection with methods that have high detection levels. Liquid Chromatography - High Resolution Mass Spectrometry (LC-HRMS) is an analytical technique that can efficiently detect drugs in biological matrices without compromising sensitivity and selectivity. Accordingly, an LC-HRMS method for the identification of 43 fentanyl analogs in equine serum was developed and validated. Application of this methodology can allow for better enforcement of anti-doping regulations for the protection of racehorses. The ability to identify fentanyl and fentanyl analogs in serum will support anti-doping measures in horse racing, as well as promote an efficient means of testing in equine drug testing labs.