Acetaminophen (APAP) overdose causes liver injury, but in some cases it is associated also with renal impairment. While several studies exist in relation to acetaminophen nephrotoxicity, no reports have been published describing intracellular changes related to APAP nephrotoxicity in vitro. Because proximal tubular cells are considered to constitute a secondary site of drug-induced injury after hepatocytes, our study's aim was to estimate the toxicity in the human HK-2 cell line. We used a range of APAP concentrations (1-10 mM) to examine toxicity in the cells (1-48 h). We evaluated cell viability using the WST-1 and LDH tests. Cells impairment was also determined by monitoring ROS production, glutathione levels. We proved that HK-2 cells are able to metabolize acetaminophen. We observed moderate impairment of cells already after 1 h of treatment based on a finding of increased ROS production and decreased cell viability. After 24 h, the results showed significant cellular impairment at all tested concentrations except for 1 mM APAP, but no glutathione depletion was found. We conclude that HK-2 cells are susceptible to acetaminophen toxicity but, unlike hepatocytes, it might be not linked to glutathione depletion.