Fluticasone propionate (FP) is an anti-inflammatory agent with topical and inhaled applications commonly used in the treatment of asthma in steroid-dependent individuals. The drug is used in racehorses to treat Inflammatory Airway Disease; this work was performed in order to advise on its use and detect potential misuse close to racing. Methods were developed for the extraction and analysis of FP from horse plasma and a carboxylic acid metabolite (FP-17βCOOH) from horse urine. The methods utilize ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in order to detect the extremely low concentrations of analyte present in both matrices. The developed methods were used to analyse plasma and urine samples collected following inhaled administration of FP to six thoroughbred horses. FP was detected in plasma for a minimum of 72 h post-administration and FP-17βCOOH was detected in urine for approximately 18 h post-administration. The results show that it is possible to detect FP in the horse following inhaled administration. Copyright © 2012 John Wiley & Sons, Ltd.