Acetaminophen (N-acetyl para amino phenol, APAP) is a widely used antipyretic and analgesic drug responsible for various drug-induced liver injuries. This study evaluated APAP-induced toxicity in isolated rat hepatocytes alongside the protective effects of silafibrate and N-acetyl cysteine (NAC). Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via the portal vein. This technique is based on liver perfusion with collagenase after removing calcium ions (Ca ) with a chelator. Cells were treated with different concentrations of APAP, silafibrate, and NAC. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarisation were measured as toxicity markers. ROS formation and lipid peroxidation occurred after APAP administration to rat hepatocytes. APAP caused mitochondrial depolarisation in isolated cells Administration of silafibrate (200 μmol L ) and/or NAC (200 μmol L ) reduced the ROS formation, lipid peroxidation, and mitochondrial depolarisation caused by APAP Cytotoxicity induced by APAP in rat hepatocytes was mediated by oxidative stress. In addition, APAP seemed to target cellular mitochondria during hepatocyte damage. The protective properties of silafibrate and/or NAC against APAP‑induced hepatic injury may have involved the induction of antioxidant enzymes, protection against oxidative stress and inflammatory responses, and alteration in cellular glutathione content. Acetaminofen (N-acetil-para-aminofenol, APAP) često je korišteni antipiretik i analgetik koji može izazvati oštećenja jetara. Na modelu izoliranih hepatocita štakora istražili smo toksične učinke APAP-a i protektivne učinke silafibrata i N-acetilcisteina (NAC). Hepatociti su izolirani iz mužjaka štakora soja Sprague-Dawley perfuzijom jetara i uvođenjem enzima kolagenaze putem portalne vene. Ta se tehnika zasniva na perfuziji jetara kolagenazom nakon uklanjanja kalcijevih iona (Ca ) kelatorom. Stanice su tretirane različitim koncentracijama APAP-a, silafibrata i NAC-a. Kao markeri toksičnosti mjereni su smrt stanica, stvaranje reaktivnih kisikovih vrsta (ROS), lipidna peroksidacija i depolarizacija mitohondrija. Primjena APAP-a u štakora izazvala je stvaranje ROS-ova i lipidnu peroksidaciju. APAP je uzrokovao depolarizaciju mitohondrija u izoliranim stanicama. Primjena silafibrata (200 μmol L ) i/ili NAC-a (200 μmol L ) smanjila je stvaranje ROS-a, lipidnu peroksidaciju i depolarizaciju mitohondrija uzrokovanu APAP-om. Utvrdili smo da je citotoksičnost APAP-a posredovana oksidativnim stresom. Nadalje, čini se da su mitohondriji ciljni stanični organeli za oštećenja hepatocita izazvanih APAP-om. Moguće je da su protektivna svojstva silafibrata i/ili NAC-a protiv APAP‑om induciranog oštećenja jetara uključivala i indukciju antioksidacijskih enzima, zaštitu od oksidativnog stresa i upalnih odgovora te promjenu razine staničnoga glutationa.