Although the influence of microbial invasion of the amniotic cavity on the development of spontaneous preterm delivery is unquestionable, the use of an invasive procedure to diagnose the status of an infection limits its clinical translation.This study aimed to use exploratory and confirmatory analyses to investigate the presence of vaginal metabolome expression of microbial invasion of the amniotic cavity in women diagnosed as having preterm labor using high-performance liquid chromatography.In 140 women with singleton pregnancies and a diagnosis of preterm labor at <34 weeks' gestation, we analyzed vaginal amino acid concentrations using high-performance liquid chromatography. Vaginal samples were collected shortly after the amniocentesis performed at admission to rule out microbial invasion of the amniotic cavity. Data were normalized for the median of all the amino acid concentrations evaluated. Microbial invasion of the amniotic cavity was defined as a positive aerobic or anaerobic amniotic fluid culture for the presence of bacteria or yeast or Ureaplasma species or Mycoplasma hominis in the mycoplasma culture or a positive polymerase chain reaction result for 16S rRNA gene sequence. Exploratory analysis was performed in half of the sample and confirmatory analysis in the other half. We compared vaginal amino acid concentrations between women with and without microbial invasion of the amniotic cavity in both cohorts. The area under the curve with 95% confidence interval values were calculated for vaginal amino acids with significant differences.In the exploratory cohort (2014-2015), 17 of 76 women (22.3%) had microbial invasion of the amniotic cavity compared with 14 of 72 (19.4%) in the confirmatory cohort (2016-2017). In the exploratory cohort, we found significantly higher amino acid concentrations of vaginal taurine, lysine, and cysteine and significantly lower concentrations of vaginal glutamate, aspartate, and the aspartate to asparagine ratio. These significant differences were confirmed in the confirmatory cohort. The area under the curve of these vaginal amino acids to predict microbial invasion of the amniotic cavity ranged between 0.72 and 0.79, with cysteine being the amino acid with the best performance with an area under the curve of 0.79 (95% confidence interval, 0.71-0.88).We found the vaginal metabolome expression of microbial invasion of the amniotic cavity in women with preterm labor and intact membranes. These findings might open the possibility to develop noninvasive diagnostic tools of microbial invasion of the amniotic cavity with the aim of selecting women who would most likely benefit from an amniocentesis for this indication.