Rationale Salinomycin is an ionophore antibiotic with potential anticancer activity. The history of its use in veterinary medicine shows large differences in species susceptibility to its toxicity. At the same time, the results of research to date suggest a correlation between the extent and pathways of ionophore biotransformation and its toxicity. The biotransformation pattern of salinomycin has not been studied so far. Methods Extracts from culture media of human hepatoma cells (HepG2) exposed to salinomycin were analysed with two mass spectrometry techniques. For the first one, micro‐liquid chromatography coupled with a quadrupole time‐of‐flight (Q‐TOF) mass spectrometer was used. In the second approach, high‐performance liquid chromatography was coupled with a hybrid triple quadrupole linear ion trap. Both experiments were operated in positive electrospray ionization mode. To identify unknown salinomycin metabolites, information‐dependent acquisition was applied. Results Metabolites identified with tandem mass spectrometry included hydroxylated, demethylated and hydroxylated–demethylated derivatives, in total 14 compounds. Using high resolution, only eight isomers of hydroxysalinomycin were detected. The efficiency of biotransformation was low, and so was the abundance of the signals; only for two metabolites did the signal exceed 1% of the salinomycin signal. The analysis of fragmentation patterns narrowed the structure combinations but the actual modification site could not be specified. Conclusions Tandem mass spectrometry was more sensitive in the identification of salinomycin metabolites in comparison to the Q‐TOF approach. Because of low efficiency of biotransformation of the applied model, the obtained fragmentation data are not sufficient to fully characterize the detected compounds. A study with more metabolically active primary hepatocytes is needed.