Acetaminophen (APAP) is the most-used over-the-counter analgesic among pregnant women. However, concerns have arisen over the safety of APAP exposure during gestation. In particular, it's been speculated that the hepatotoxic metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), forms in the brain after maternal use of therapeutic APAP doses and leads to neurodevelopmental disorders (NDDs). However, APAP metabolism in the brain is understudied. Here, we tested the hypothesis that NAPQI can be generated in the brain by overdosing BTBR T+Itpr3tf/J (common model of the NDD autism) and C57Bl/6J mice with APAP and measuring glutathione loss and APAP-protein adducts as two of the best markers of NAPQI available. Despite glutathione depletion and adducts in the liver, we saw none in the brain. We conclude NAPQI is unlikely to contribute to the pathophysiology of NDDs. IMPACT STATEMENT: It has been hypothesized that NAPQI formation in the brain provides biological plausibility for the purported link between APAP and NDDs. Our results cast doubt on that hypothesis.