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Naphthenic acids present formidable challenges for the petroleum industry and are a growing concern in the aquatic environment. For example, these acids are responsible for corrosion of refinery equipment, leading to the incurrence of additional costs to the consumer, and are toxic to aquatic wildlife, making disposal and remediation of contaminated waters and sediments a significant problem. The detection and characterization of naphthenic acids is therefore of considerable importance. Fourier transform ion cyclotron resonance mass spectrometry is presented as a technique with inherently ultra-high mass accuracy and resolution, affording unequivocal assignments. The suitability of the technique for environmental applications is demonstrated to characterize two different commercial mixtures of naphthenic acids and one oilsands tailings pond sample.