During the oil sand hot water extraction process, naphthenic acids are extracted from the bitumen into the oil sands process water, which leaves the water acutely and chronically toxic to aquatic biota. Because these acids can have toxic effects even at relatively low concentrations, depending on their composition and age, it is important to have analytical techniques sensitive enough to quantify the amounts of naphthenic acids in the aqueous environment. In this study, a new HPLC-MS/MS method was developed to separate and analyze mixtures of naphthenic acids. Using this method, inorganic salts are separated from the acids without any sample pre-treatment, such as an extraction step. Different naphthenic acids give characteristic retention times due to their structures, thus enabling measurements of the individual concentrations. Adsorption characteristics of certain naphthenic acids were explored using the new analytical method. It is expected that this analytical method could be employed to determine the fate and stability of individual naphthenic acids in the environment and thus provides insight to potential environmental impacts of oil sands processing.