Naphthenic acid fraction compounds (NAFCs) are naturally present in the oil sand. These compounds become integrated into the oil sands process-affected water (OSPW) during the bitumen extraction process. NAFCs have been identified as causing toxicity in the OSPW to aquatic organisms. Water treatment technologies that are largely passive, such as constructed treatment wetlands, are a sought-after technology for the degradation of NAFCs in aquatic environments, partly because of their low energy intensity. However, it can be challenging to accurately assess the performance, regarding decreased NAFC concentration and biodegradation characteristics in water samples that have been exposed to such systems. This is due to interferences of biological products such as fatty acids and humic-like materials, which may give false-positive information 911 NAFCs estimation with conventional analytical sample cleanup methods such as liquid liquid extraction (LLE). It is recognized that this same issue exists when attempting to characterize NAFCs in natural wetlands for environmental monitoring purposes and, therefore, an analytical method that can remove background interferences in water samples is desirable on several fronts. Studies were thus conducted to develop and compare methods for NAFC isolation in an experimental wetland setting. A controlled greenhouse experiment was conducted with sedge (Carex aquatilis), bulrush (Schoenoplectus acutus), and. cattail (Typha latifolia) grown in OSPW. Two methods the Isolute Biotage ENV+ SPE method and a new weak anion exchange (WAX SPE) were assessed for their ability to isolate, clean up, and concentrate NAFCs in OSPW and municipal tap water (control) that were exposed to samples of plants and associated microbes. Negative-ion-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) data revealed that WAX SPE method has better relative enhancement (5%-50%) of O-2 classes in OSPW exposed to wetland plants, compared to ENV+ SPE method. The WAX SPE method is a good candidate for the isolation of organic compounds in complex environmental matrices and supports the development of analytical protocols for isolation and characterization of NAFCs. Compound classes from negative-ion BSI-FT-ICR-MS data were further probed using principal component analysis (PCA) to evaluate the NAFCs that are potential indicators of efficiency of engineered wetlands for monitoring in future wetland studies. Given the PCA results, future wetland NAFC degradation investigations should target O-2 classes for detailed evaluation of the performance of treatment systems, or measurement of the fate and distributions of NAFCs in natural wetlands exposed to OSPW.