This pilot study investigated the possibility that metabolomic differences exist in second trimester of women delivering at term (≥37 weeks, n = 216) and preterm (≤35 weeks, n = 11). For this retrospective study, biobanked AF samples underwent near-infrared (NIR) spectral analysis using wavelengths from 700 to 1050 nm. Spectral data was compressed then optimized by multilinear regression to create a calibration model. The resultant model was able to classify term and preterm births based on differing AF metabolomic profiles with a sensitivity and specificity of 100%. When groups were classified using a prematurity index (PI), there was a statistical difference (P < 0.001) between the predicted preterm group (PI 0.77 ± 0.08) and the term group (PI 1.00 ± 0.02). In conclusion, the 2nd trimester AF samples showed distinct differences in metabolomic profiles between patients delivering preterm as compared to those at term in functional groups related to proteins, carbohydrates, fats, polyols, and water.