Abstract: AimTo investigate the metabolic profile of roxithromycin in dogs and the effects of oral and intravenous administrations on the metabolism of roxithromycin. MethodsLiquid chromatography-tandem mass spectrometry (LC-MSⁿ) was used for separation and analysis of roxithromycin and its metabolites in dog bile after an oral dose or intravenous dose of roxithromycin. The metabolites were identified by comparisons of their mass spectra and LC behaviors with the references. ResultsTotally 13 metabolites were detected in dog bile, including N-demethylated derivatives, N, N-didemethylated derivatives, O-dealkylether derivatives, decladinose derivatives, and the geometric isomers of parent drug and its metabolites. ConclusionRoxithromycin underwent 4 metabolic pathways in which geometric isomerization and decladinose metabolism were found to be markedly different between the two administration routes.