EC-18 (i.e., 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), an active ingredient in Rockpid®, has been reported to be useful in controlling various types of inflammations, particularly those caused by neutropenia. Although this product was originally approved as a functional food in Korea, it is currently in phase II clinical trials for use in managing the severe chemotherapy-induced neutropenia in patients with advanced breast cancer who are receiving intermediate febrile neutropenia risk chemotherapy. The objective of this study was to develop a rapid, sensitive method for the determination of EC-18 in rat and mouse plasma and to evaluate the applicability of the assay in pharmacokinetic studies. EC-18 was extracted with MeOH from rat and mouse plasma samples, and the extract directly introduced onto an LC–MS/MS system. The analyte and EC-18-d3, an internal standard, were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 635.4 → 355.4 for EC-18 and 638.4 → 338.4 for the internal standard, respectively. The lower limit of quantification (LLOQ) was determined at 50 ng/mL, with an acceptable linearity in the range from 50 to 10,000 ng/mL (r > 0.999) for both matrices. Validation parameters such as accuracy, precision, dilution, recovery, matrix effects and stability were found to be within the acceptance criteria of the assay validation guidelines, indicating that the assay is applicable for estimating EC-18 in concentrations in the range examined. EC-18 was readily determined in plasma samples for periods of up to 8 h following an intravenous bolus injection of 1 mg/kg in rats and at 5 mg/kg in mice, respectively, and up to 24 h following the oral administration of 2000 mg/kg in mice. The findings indicate that the current analytical method is applicable for pharmacokinetic studies of EC-18 in small animals.