Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 μM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI–glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K_m value 130 μM (95% confidence interval = 63–210 μM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.