Bis(2,4-di-tert-butylphenyl)Phosphate (bDtBPP) leaches out of polyethylene films used by the biopharmaceutical industry in single-use systems (SUS) for the culturing of drug producing cell lines. Previous studies found bDtBPP (0.025 - 0.110 mg/L) negatively affects Chinese hamster ovary (CHO) cell growth and productivity. Less information is known about the potential early stages of subtle pre-lethal cytotoxic effects of bDtBPP. This study aimed to investigate the pre-lethal cytotoxic effects in CHO-K1 cells of bDtBPP (0.005 - 0.25 μg/ml) at process relevant concentrations following 2, 24 and 48 h exposure, using high content analysis to investigate multiple pre-lethal cytotoxicity markers. After 48 h exposure, bDtBPP (0.005 - 0.25 μg/ml; P ≤ 0.001) increased nuclear intensity. A dose- and time-dependent reduction in mitochondrial mass was seen after exposure to bDtBPP. Reactive oxygen species increased after 2 h exposure to 0.25 μg/ml bDtBPP, 24 and 48 h exposure to 0.05 - 0.25 μg/ml bDtBPP (P ≤ 0.01 and P ≤ 0.001). BDtBPP induced subtle pre-lethal cytotoxic effects on CHO-K1 cellular health. This study highlights the cellular health benefits of the biopharmaceutical industry switching to alternative SUS plastics which do not leach bDtBPP, which may enhance CHO-K1 cell productivity.