Naphthenic acids (NAs) are complex mixtures of naturally occurring acyclic and cyclic aliphatic carboxylic acids, with general formula Cn H 2n+Z O2 . They are responsible for the toxicity of the water in the tailings ponds of the oil sands mining industry. In this work, negative ion electrospray ionization mass spectrometry (ESI-MS) has been proved to be a reliable and sensitive method for the characterization of NAs. ESI-MS was shown to be superior to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) for the quantification of congeners using a standard addition procedure. Direct analysis of NAs from the tailings pond water was unsuccessful due to background ions' interference. Partial separation of the congeners was achieved with anion exchange chromatography, and the fractions were subjected to ESI-MS analysis and toxicity assessment using the Microtox assay. This suggested that the Z = -4 family was responsible for a significant part of the aquatic toxicity of NAs.