Nephrotoxicity is a serious side effect of many drugs due to the kidney being one of the major sites for their excretion. In vitro models of nephrotoxicity are therefore paramount in drug development process, which would be used to detect early stages of drug-induced toxicity. This study investigates the use of human and rat primary proximal tubule cells as in vitro models of nephrotoxicity. Human and rat proximal tubule cells (PTCs) were isolated and cultured from cortical tissues of the kidney. The cells form highly polarised monolayers that were exposed to polymyxin B, gentamicin and cisplatin – all well-known nephrotoxins. Cell viability and LDH-based cell death were measured, alongside the production of renal toxicity biomarkers, KIM-1, NGAL and clusterin. As expected, cell viability of the monolayers after polymyxin B, gentamicin and cisplatin challenge was deceased. The proportion of cells alive was dependent on the concentration and period of exposure of the nephrotoxin. For example, the cell viability of rat PTCs treated with gentamicin for 48h decreased by 43% than control cells (P < 0.01). Higher levels of KIM-1, NGAL and clusterin secretion were detected when human and rat PTCs were treated with nephrotoxins. All three biomarkers were predominately secreted across the apical membrane of the PTCs monolayers. For instance, KIM-1 level across the apical membrane was 0.9±0.05 ng/ml, which was significantly higher than across the basolateral membrane at 0.35±0.04 ng/ml. The levels of biomarkers secretion were also found to be dependent on concentrations of nephrotoxin and period of exposure. For example, rat PTCs treated with 250 µg/ml polymyxin B for 24h produced 5.5±0.7 ng/ml of KIM-1, which increased to 19±0.1 ng/ml with 48h challenge, an increase of more than 3-fold (P <0.01). The mechanisms of toxicity were also investigated by pre-treating the PTCs with supposedly nephron-protectant, rosuvastatin, cilastatin and cimetidine, prior to nephrotoxin exposure. While 50 µM rosuvastatin did not change the cell viability nor KIM-1 expression levels in human and rat PTCs, the pre-treatment of rosuvastatin with polymyxin B increased cell viability by 15 % when compared to polymyxin-B only treatment. Similarly rat PTCs treated with 250 µg/ml gentamicin and 40 µM cilastatin showed KIM-1 at levels of 7.5±0.3 ng/ml, compared with 11.5±1.2 ng/ml in cells treated with only gentamicin, giving a decrease of 35 % (P < 0.05). These data suggest mechanisms of the aminoglycoside uptake could be via megalin/cubilin receptors, which were inhibited by rosuvastatin and cilastatin. These data show the utility of human and rat PTCs as in vitro models for the study of nephrotoxicity, and their potential in elucidating the mechanisms of action of polymyxin-B, gentamicin and cisplatin induced toxicity.