Drug-induced liver injury is one of the main causes of drug non-approval and drug withdrawal by the Food and Drug Administration (FDA). Acetaminophen (APAP) is a widely used non-steroidal anti-inflammatory drug for treating fever and headache. APAP is considered safe at therapeutic doses; however, there have been reports of acute liver injury following the administration of APAP. To explore APAP hepatotoxicity and its mechanisms, we designed and synthesized a new click chemistry probe, APAP-P1, in our current study. We introduced the PEG-azide probe linker into the acetyl group of acetaminophen. First, we evaluated the probe toxicity in HepaRG cells and found that it still retained hepatotoxicity. We also found that this probe APAP-P1 can be metabolized by HepaRG cells. This demonstrated that the APAP-P1 probe still kept its metabolism characteristics. Using this probe, we pulled down its potential targets and . APAP can directly target TrxR1; thus, we tested for this interaction by Western blotting of pull-down proteins. The results showed that APAP-P1 can pull down TrxR1 and .