Oxylipins, a family of oxygenated natural products derived from polyunsaturated fatty acids (PUFAs), play crucial roles in various physiological processes. Evaluating their levels in vivo helps to reveal their roles in health and disease. Because of the numerous isomers of oxylipins, it is essential to develop efficient and precise analytical methods for their identification and quantification. The objective of this study is to establish a quantitative method for oxylipin analysis and its application to the assessment of oxylipins in children's plasma, with potential implications for diagnostic use in pediatric populations. A liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed to quantify 64 oxylipins and four precursor PUFAs within 36 min. The limits of quantification ranged from 0.25 to 50 pg, with most analytes showing recoveries and matrix effects between 85 and 110% and between 90 and 110%, respectively. Intra- and inter-day precision values were within 15%. The established method was applied to plasma samples from children aged 9-12 years (boys = 181; girls = 161) in Hokkaido, Japan, to assess the relation between plasma oxylipin and PUFA levels and age, sex, and body mass index. There was no significant correlation between oxylipin levels and age, sex, or body mass index. However, among the PUFAs, boys had higher eicosapentaenoic acid and arachidonic acid levels than those of girls, with a significant increase in eicosapentaenoic acid levels in the overweight group compared with those in the underweight group. We successfully developed a simple and highly selective method for the analysis of oxylipins in preadolescent children's plasma samples. Thus, this study provides a foundation for broader application of the developed method to different biological samples in future studies.