The aim of current study was to identify human phase I metabolites of omeprazole using mass spec- trometry. An in-vitro assay was performed to generate metabolites by human liver enzymes. Fragmenta- tion pathway of omeprazole was studied for the structure elucidation of metabolite by MS/MS and MS 3 method using previously detected fragments. The base peak of omeprazole with m/z 198 was used for a precursor ion scan to detect possible metabolites containing m/z 198 as a fragment. Additional metabo- lites should be detected by the loss of a typical neutral group using m/z 148 for the offset of a neutral loss scan. The detected precursor ions ( m/z 316, 330, 332, 346, 362) were analyzed by enhanced product ion scan in order to receive a mass spectrum for the comparison with the fragmentation of omeprazole. The three reactions (oxidation, reduction of the Sulphur and demethylation) were observed as phase I reactions.