A simple and sensitive liquid chromatography method with absorbance or mass spectrometry detection was developed and validated for the determination of lansoprazole, lansoprazole sulfone, and 5-hydroxylansorpazole in human serum with omeprazole as an internal standard. Analytes were, after an extraction step with a mixture of isopropyl alcohol: dichloromethane (2:8, v/v), separated in a gradient elution (acetonitrile: 20 mM ammonium formate) and detected at 280 and 300 nm or by monitoring selective reactions in a triple quadrupole mass analyzer. The limits of detection were, for all analytes, below 2 ng/mL and 3 pg/mL in the LC UV and LC MS method, respectively. The combined linear dynamic range exceeded 5 orders of magnitude. The method was successfully applied in the assessment of CYP2C19 and CYP3A4 metabolic phenotypes in ST-elevated myocardial infarction patients receiving novel anticoagulant treatment.