The aim of the study was to evaluate the plasma profile of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), as well to analyze the relationship of Omega 6/Omega 3 ratio with anthropo-metric parameters and insulin resistance markers. Material and methods: Plasma levels of free fatty acids (FFAs) were measured using a high-throughput LC-MS AB Sciex4600 in 202 children (127 obese and 75 non-obese), age and sex-matched. Lipid and glucose profiles were assessed with current laboratory methods, while insulin resistance and beta-cell function were evaluated using HOMA-IR and HOMA-β respectively. Results: In obese children, AA and AA/(DHA+EPA) ratio were significantly higher regardless of age and gender. In the lowest quartile of DHA, there was a clear trend for insulin resistance, with plasma insulin level, HOMA-IR, and HOMA-β significantly higher compared to the highest quartile of DHA. After adjustment for age and gender DHA remains a negative predictive factor for insulin resistance. Waist-to-height ratio (WHtR), a marker of visceral obesity was higher in children with a higher AA/(DHA+EPA) ratio. Conclusions: In obese children, the AA is higher in concordance with insulin resistance. Additionally, children with a higher AA/(DHA+EPA) ratio have greater BMI, fat mass, waist circumference, and WHtR, important indicators of central adiposity, and cardio-metabolic disorders. LC/MS is a versatile tool for Omega ratio assessment, especially in children where the sample size is a limiting factor for metabolic and nutrition evaluation.