In equine doping control, screening methods are needed to detect diverse compounds at varying concentrations in a short period of time. High-resolution mass spectrometry offers enhanced specificity and sensitivity which provides accurate screening of doping substances. Different classes of doping agents, such as barbiturates, hypoxia-inducible factors (HIFs), nonsteroidal anti-inflammatory drugs (NSAIDs), and prolyl-hydroxylase inhibitors (PHIs), were extracted from equine plasma by liquid-liquid extraction with methyl tert-butyl ether and 1 M phosphoric acid. An ACE 5 C18 column (75 x 2.1 mm) was used with mobile phases of 5 mM ammonium formate and acetonitrile. A high-resolution mass spectrometry (HRMS) screening method for detection of more than 60 compounds was developed using a Q-Exactive Plus mass spectrometer operating in negative ion mode. A full scan/dd-MS2 method was used to acquire HRMS/MS data with a resolution of 70,000 for full scan and 17,500 for dd-MS2 scan. A HRMS/MS compound database and spectra library were developed and optimized for each compound to improve specificity and sensitivity. TraceFinder software version 5.1 was used for data acquisition, processing, and data review. The total analysis time was 7.5 minutes with retention times ranging from the earliest at 2.5 min to the latest at 5.8 min. Stable retention times were observed from run to run. The LC method provided separation of substances with sharp, symmetrical peak shapes with minimum tailing or broadening. Accurate mass, isotopic pattern, MS/MS spectra, product ions, and retention time were used to identify a suspect sample. The limit of detection of each compound was validated using retention time and accurate mass.