Canada is among the world’s leading producers and exporter of diluted bitumen (dilbit). Despite this, there is a lack of understanding surrounding the effects of dilbit spills in freshwater ecosystems. F2-isoprostanes (F2-IsoPs) are reliable biomarkers of oxidative stress, one of dilbit’s toxicity pathways in fish, however they are seldom studied in fish. This thesis describes the development of a non-invasive method for measuring F2-isoprostanes in the epidermal mucus of fish to measure oxidative stress in fathead minnows (Pimephales promelas) exposed to dilbit. An HPLC-MS/MS method was first developed and validated using Northern pike mucus (Esox lucius), and used to successfully detect F2-IsoPs in lake trout (Salvelinus namaycush) angled at the IISD-Experimental Lakes Area in Northwestern Ontario, proving these compounds are present at detectable concentrations in wild fish mucus. The relative concentrations of F2-IsoPs were then examined in tissues of rainbow trout (Onchorynchus mykiss) exposed to acute physical stress. F2-IsoP concentrations were also compared to total glutathione (GSH) and the GSH redox status in this study, finding a possible link between F2-IsoPs and GSH in gills and evidence of a mild oxidative stress response in physically stressed trout. The effect of dilbit on epidermal mucus and liver F2-IsoP concentrations was then examined in juvenile fathead minnows exposed for 7 days to 0-30% dilbit solutions, or water accommodated fraction (WAF). F2-IsoP concentrations did not differ significantly between any dilbit treatments: however this lack of response may have been caused by low uptake of polycyclic aromatic compounds. Liver and mucus F2-IsoPs were positively correlated in the control group and negatively correlated in the 30% WAF treatment. Finally, the developed method was field-tested in the Freshwater Oil Spill Remediation Study (FOReSt), a collaborative project aiming to determine the fate and behaviour of dilbit in a freshwater shoreline environment. Adult fathead minnows were exposed to environmentally relevant concentrations of weathered dilbit for ~75 days in shoreline enclosures at the IISD-ELA. Mucus F2-IsoPs were quantified in fathead minnows and finescale dace (Phoxinus neogaeus). Concentrations were not significantly different between treatments, suggesting dilbit exposure did not cause a lasting oxidative stress response in either species.