Down Syndrome is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. Metabolomics is identification and quantification of small-molecule metabolites (molecular weight <1000 Da) in tissues, cells and physiological fluids within a certain period time. Metabolites are intermediate products of various types of biochemical reactions that participate in bonding metabolic pathways. In this study, metabolites such as 2-Hydroxybutyric acid, 3-Hydroxybutyric acid, beta-Hydroxyisovaleric acid, Uracil, Glutamic acid, Maltose and Melezitose were chosen as the possible determinants/markers for the prenatal screening of Down Syndrome. Quantitative analysis of the metabolites conducted by GC-MS method using 5 % phenyl / 95 % dimethylpolysiloxane (30 m x0.25 mm, 0.25 mu m film thickness) capillary column. The oven temperature was held constant at 60 degrees C for 1 min and ramped at 10 degrees C /min to 200 degrees C then ramped at 30 degrees C/min to 320 degrees C and hold for 6 min before cool-down, as helium mobile phase and flow rate of 2.8 mL/min and adding Myristic acid-d27 as an internal standard. Our method was validated by parameters of system suitability, stability, linearity, sensitivity, accuracy, precision, selectivity, robustness and ruggedness. The developed and validated method was applied to plasma samples taken from pregnant women with Down Syndrome (study group) and euploid fetuses (healthy group). The levels of these seven metabolites are statistically different (p < 0.05 for all) between the groups. It can be concluded that these relevant metabolites might be used for the prenatal screening of Down Syndrome. (C) 2020 Elsevier B.V. All rights reserved.