Arnica montana preparations are topically utilized to treat injuries and inflammations, as well as rheumatic muscle and joint complaints. The sesquiterpene lactones helenalin as well as 11α,13-dihydrohelenalin and their esters are known to be the active compounds in Arnica preparations. Leishmaniasis is one of the 20 communicable diseases currently classified by WHO as neglected tropical diseases. In recent studies, an ethanolic tincture of A. montana flowers effectively cured cutaneous leishmaniasis (CL) in a golden hamster model. To use Arnica preparations on open wounds such as CL lesions, absorption, distribution, metabolism and excretion of Arnica ingredients have to be analysed. Experiments on in vitro metabolism of the sesquiterpene lactone 11α,13-dihydrohelenalin acetate, were carried out. Phase I metabolism was investigated with pig liver microsomes and an NADPH-regenerating system. Afterwards, metabolites were detected and identified with UHPLC/ESI QqTOF MS/MS analysis. The Phase I reactions were stopped at different time points to gain time dependent information about the development of metabolites. Difference chromatograms of the UHPLC/ESI QqTOF MS chromatograms of the samples and matrix controls were calculated with MetaboliteDetect 2.0 (Bruker Daltonics). The detected metabolites were formed by addition of glutathione, expoxidation and addition of water to various positions.