In vitro models, including the widely used PC12 cell line, can increase insight into cellular and molecular mechanisms underlying neurodegenerative processes. An important determinant for the vulnerability of cells for chemical insults may be the endogenous level of oxidative stress. To test this hypothesis, we induced different degrees of cellular stress in PC12 cells by altering their ROS production using dexamethasone, l-dihydroxyphenylalanine (l-DOPA) and iron. These different PC12 models were subsequently used to investigate whether the degree of cellular stress could increase their susceptibility to environmental pollutants. The characteristics of these stressed PC12 cell subtypes and their vulnerability to the reference pesticide rotenone were investigated using a combination of biochemical (oxidative stress, cell viability, and α-synuclein expression) and functional (fluorescent calcium imaging) assays. Our combined data demonstrate that chemically-induced stress in PC12 cells increases the production of reactive oxygen species (ROS) and alters calcium homeostasis and α-synuclein expression. Moreover, l-DOPA and FeSO4 pre-treated PC12 cells show increased vulnerability to rotenone-induced cytotoxicity. These chemically-stressed cell models may therefore prove valuable to investigate how increased cellular stress influences neurotoxic outcome, for example in case of mixture toxicity.