Using Fourier transform infrared (FT-IR) spectroscopy we investigated the water content of SAE 15W–40 diesel engine lubricating oil at various levels of contamination to establish instrument calibration standards for measuring water contamination in used or in-service engine oil by the standards of ASTM International. Since some known additives in consumer grade engine oil possess slightly hydrophilic properties, this experiment avoided changing the sample matrix with supplemental additives, such as adding surfactants, to achieve homogeneity of the original sample. The impact of sampling time after contamination on the spectral absorption signature was examined in an attempt to improve the accuracy of water contamination quantification and determine if water-soluble potassium bromide (KBr) windows were suitable for analyzing water in oil emulsions. Analysis of variance (ANOVA) modeling and limit of detection calculations were used to predict the ability to discriminate contamination levels over time. Our results revealed that the amount of water concentration in engine oil could be misinterpreted depending on the timing of the FT-IR measurement of the calibration standard after initial water contamination. Also, KBr windows are not sufficiently etched due to the limited window interaction with water molecules within micelles of emulsions to alter FT-IR spectral signatures.