Background: Horse urine is the medium of choice for the implementation of metabolomic approaches aimed at improving horse doping control. However, drug analysis in this biofluid is a challenging task due to the presence of large amounts of interfering compounds. [br/] Methodology & Results: A comparative study of sample preparation has been conducted to evaluate five sample-preparation methods, namely acetonitrile precipitation, proteinase K hydrolysis, membrane filtration and sample dilution with water by factors of five and 20, for metabolome analysis using liquid chromatography coupled to high resolution mass spectrometry. Assessment was performed at both global and targeted levels, by using a few thousand features obtained from peak detection software, and internal standards and 100 annotated or identified metabolites. [br/] Conclusion: By considering the number of detected signals, their intensity and their detection repeatability, acetonitrile precipitation was selected as the most efficient sample-preparation method for the analysis of horse urine metabolome in liquid chromatography coupled to high resolution mass spectrometry conditions.