The development of biomarkers of chemical exposure and effect in threatened wildlife is challenging because traditional methods for biomarker discovery that involve in vivo testing or destructive sampling cannot be utilised. These challenges have long since been acknowledged and the development of non‐destructive methods for the detection of biomarkers in wildlife has been examined for many decades. However, despite these efforts, progress in this field has been slow and the development or confirmation of non‐destructive biomarkers of exposure in threatened wildlife has been limited. The usefulness of cell lines along with non‐targeted omics has not been well explored in this context despite the potential for these methods to greatly enhance non‐destructive biomarker development. In order to explore the usefulness of global protein expression of primary cells cultured from wildlife, we optimised exposure and extraction methods and aimed to initially examine the effect of time and dose on global protein expression. Primary green sea turtle (Chelonia mydas) skin cells developed in our lab were exposed to two contaminants known to accumulate in sea turtles ‐ a polychlorinated biphenyl (PCB153) and perfluoronanoic acid (PFNA). Three environmentally relevant concentrations were chosen for exposure (1 μg/L, 0.1 μg/L, and 0.01 μg/L) and cells were exposed for both 24 or 48 hours. The global protein expression was then quantified using Sequential Windowed Acquisition of all Theoretical Fragment Ion Mass Spectra (SWATH‐MS) resulting in over 1000 unique protein identifications. Our results show that a large number of proteins are significantly differentially expressed when sea turtle primary cells are exposed to chemical contaminants. Over 700 proteins were differentially expressed by cells under exposure conditions and time and concentration had significant effects on overall differential expression as well as on the expression if individual proteins. Most significantly, a biomarker of PCB exposure that has previously been identified in sea turtles (superoxide dismutase) was expressed by the skin cells exposed to PCB indicating that these methods can potentially reflect biomarkers measured in whole organisms. Overall, the results from this study provide insight into the effects that time, dose and treatment have on global protein expression of green sea turtles cells, as well as preliminary evidence for the usefulness of non‐targeted proteomics for biomarker discovery. This indicates the potential of the methods described here to support higher throughput confirmation of biomarkers of exposure and effect while reducing the need for invasive sampling on threatened wildlife.