The purpose of this study was to develop a reliable model for the calculation of gestational age (GA) in second and third trimesters with the use of amniotic fluid (AF) metabolite profiles that were determined by magnetic resonance spectroscopy.High-resolution (11.7 T) ex vivo magnetic resonance spectroscopy was performed on 95 AF samples (mean, 31.7 weeks; range, 15.6–39.9 weeks). GA was determined by last menstrual period or first-trimester ultrasound scanning. Concentrations of 21 AF metabolites were measured with automated techniques. Metabolite concentrations, inverses, natural logs, and squares were entered as predictive variables in a stepwise linear regression model.The following formula was derived: GA = 64.922 – (14.456 × alanine) + (4.965 × natural log [creatinine]) – (0.931 × glucose) – (5.202 × valine). This model fit the data with an R 2 value of 0.926. Average error among all samples was ±1.75 weeks (SD, ±1.43 weeks), for the second trimester was ±2.21 weeks (SD, ±1.78 weeks), and for the third trimester was ±1.59 weeks (SD, ±1.26 weeks).Statistical modeling accurately predicted GA with amniotic fluid metabolite profiles that were obtained by magnetic resonance spectroscopy, which may represent a significant improvement over conventional ultrasound dating in the third trimester. Future studies should compare these techniques directly.