There is a significant need to evaluate the therapeutic potential of natural products and other compounds purported to be hepatoprotective. Acetaminophen-induced liver injury, especially in mice, is an attractive and widely used model for this purpose because it is both clinically relevant and experimentally convenient. However, the pathophysiology of liver injury after acetaminophen overdose is complex. This review describes the multiple steps and signaling pathways involved in acetaminophen-mediated cell death. The toxicity is initiated by the formation of a reactive metabolite, which depletes glutathione and binds to cellular proteins, especially in mitochondria. The resulting mitochondrial oxidant stress and peroxynitrite formation, in part through amplification by c-jun-N-terminal kinase activation, leads to mitochondrial DNA damage and opening of the mitochondrial permeability transition pore. Endonucleases from the mitochondrial intermembrane space and lysosomes are responsible for nuclear DNA fragmentation. Despite the oxidant stress, lipid peroxidation is not a relevant mechanism of injury. The mitochondrial dysfunction and nuclear DNA damage ultimately cause oncotic necrotic cell death with release of damage-associated molecular patterns that trigger a sterile inflammatory response. Current evidence supports the hypothesis that innate immune cells do not contribute to injury but are involved in cell debris removal and regeneration. This review discusses the latest mechanistic aspects of acetaminophen hepatotoxicity and demonstrates ways to assess the mechanisms of drug action and design experiments needed to avoid pitfalls and incorrect conclusions. This review should assist investigators in the optimal use of this model to test the efficacy of natural compounds and obtain reliable mechanistic information.