An in-depth experimental study of the matrix effect of antifreeze (ethylene glycol) and water contamination of engine oil through FT-IR spectroscopy. With a comparison of the percent by volume concentration of contaminated fresh 15W-40 engine oil, there appeared to be a noticeable reduction in the O–H stretching signal in the infrared spectrum when ethylene glycol based antifreeze was included as a contaminant. The contaminants of distilled water, a 50/50 mixture of water and commercial ethylene glycol antifreeze, and straight ethylene glycol antifreeze were compared and a signal reduction in the O–H stretch was clearly evident when glycol was present. Doubling the volume of the 50/50 mixture as compared to water alone still resulted in a weaker O–H stretching signal. The possibility that this signal reduction was due to the larger ethylene glycol molecule having fewer O–H bonds in a given sample size was eliminated by comparing samples with the same number of O–H bonds per unit volume. The strong hydrogen bonding between that of water and glycol appeared to reduce the O–H stretching signal, even after comparing the different sample types at concentrations with the same number of O–H bonds per unit volume. Tukey’s highly significant difference was used to show that samples of the 50/50 mixture and straight glycol were not reliably distinguishable from one another when comparing the same number of O–H bonds per unit volume but readily distinguishable from that of water as the lone contaminant.