Objective: To compare plasma free amino acid (FAA) and carnitine levels in pregnant women with and without hydatidiform mole (HM). Methods: This prospective study was conducted in patients admitted to Harran University Medical Faculty Hospital Obstetrics Clinic between January and November 2018. Twenty-three pregnant women with HM, and 24 healthy pregnant women as controls were enrolled in the study. The venous blood samples were collected, and, FAA and carnitine concentrations were measured in plasma using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Results: The levels of alanine, arginine, and valine from the 14 amino acids examined were significantly lower in the HM group than in the healthy group (475.46 ± 103.55 µmol/L vs 556.99 ± 105.16 µmol/L, p = 0.019; 428.87 ± 138.91 µmol/L vs 547.11 ± 140.20 µmol/L, p = 0.009; and 290.96 ± 97.10 µmol/L vs 358.61 ± 75.40 µmol/L, p = 0.03; respectively). In addition, several carnitines, C8DC, C16:1, and C18, of the 27 carnitines examined were significantly higher in the HM group than in the control group (0.07 ± 0.06 µmol/L vs 0.03 ± 0.01 µmol/L, p = 0.021; 0.18 ± 0.09 µmol/L vs 0.10 ± 0.06 µmol/L, p = 0.03; and 0.07 ± 0.02 µmol/L vs 0.03 ± 0.01 µmol/L, p = 0.021; respectively). Conclusion: This study demonstrated that a decrease in some plasma FAAs and an increase in some plasma carnitine levels might be effective in the pathogenesis of HM.