Naphthenic acids (general formula CnH2n+ZO2) are water-soluble, toxic compounds found in petroleum and bitumen. Some of the current methods for detecting these acids in waters depend on measuring the presence of the carboxylic acid functional group, and therefore many of these methods also detect naturally occurring carboxylic acids that are not naphthenic acids. We report a procedure that includes liquid–liquid extraction, cleanup, and derivatization to form t-butyldimethylsilyl esters prior to gas chromatography–mass spectrometry (GC–MS) analysis. Using low- and high-resolution MS to detect the ion C15H27O2Si+ (nominal m/z = 267) is an excellent indicator of the presence of naphthenic acids at concentrations ≥10 μg L−1.