Perfluorocarbons (PFCs) are chemicals that have many useful properties including the ability to carry large amounts of dissolved oxygen. PFCs are compounds comprised of a carbon-based backbone that are partially to fully fluorinated. They have been developed as potential therapeutic agents as blood substitutes for the treatment of hypovolemic shock. Due to their ability to carry oxygen with high efficacy and capacity, PFCs may be misused as doping agents in equine athletes to enhance performance. PFCs are not monitored by most extraction and instrumental analysis approaches routinely utilized in racing chemistry laboratories. Accordingly, a method to extract and detect PFCs including, perfluorohexane, perfluorooctane, trans-1,2-bis(perfluoro-n-butyl)ethene, perfluoro-tert-butylcyclohexane, perfluorodecalin, perfluoromethyldecalin, perflubron, perfluoromethylcyclopiperidin, 1,8-dichloroperfluorooctane, perfluorodecyl bromide, bromopentafluorobenzene in equine serum using headspace gas chromatography mass spectrometry (HS-GC-MS) was developed and validated. The limits of identification ranged from 0.5 to 25 μg/mL depending upon the analyte. Intra- and inter-day accuracy ranged from 86 to 144 % at a lower QC level and from 73 to 120 % at a higher QC level. Intra- and inter-day precision ranged from 3 to 8 % at a lower QC level and from 4 to 12 % at a higher QC level. The stability of the targeted PFCs in serum stored in serum separator blood tubes was assessed at room temperature, 2-8 ℃, and at -20 ℃ at various timepoints with modest reductions observed following storage at room temperature or with refrigeration while minimal losses were observed with storage at -20 ℃. These data indicate that the detection of PFCs in equine serum can be accomplished and utilized using HS-GC-MS allowing for the potential routine analysis of PFCs in horseracing laboratories.