In recent times, additional pathways involved in the regulation of the myometrium have been suggested. This also holds true for the effect of drugs such as oxytocin (OT) and -adrenergic agonists on the myometrium. Knowledge of these additional pathways will certainly prove useful in designing better therapies for pathologies of the myometrium. This study was therefore aimed at investigating the possibility of other pathways involved in the activities of both OT and ritodrine (RIT; a -adrenergic agonist) in the myometrium by utilizing metabolomics and bioinformatics. High-resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy coupled with functional uterine assays were used for an innovative assessment. In vitro pharmacological assay of OT (1 nmol/L) and RIT (0.1 nmol/L) on isolated mice uteri mounted in 3 mL organ baths was performed. Mice uteri, treated with OT or RIT, as well as the physiological buffer in which the uterine tissues were immersed, were rapidly collected and analyzed using HRFTMS, proton (H-1)-NMR, and bioinformatics. Resulting data were analyzed via pairwise chemometric comparison models, with P .05 considered statistically significant. In addition to previously known metabolites, nicotinamide adenine dinucleotide, -aminobutyric acid, and sphingosine were significantly associated with the activity of OT, whereas the activity of RIT was associated with a downstream involvement of prostaglandin F-1 and phosphatidylinositol signaling. These findings add evidence to the reports on additional regulation of myometrial activity by these drugs and suggest newer pathways for therapeutic manipulation.