Objective: The objective of this study is to evaluate diagnostic accuracy of transperineal ultrasound assessment compared to speculum examination by using placental alfa-microglobulin (PAMG)-1 tests as reference.Methods: This was a prospective observational study conducted in Ankara University Hospital. Women with early and late preterm pregnancies with suspected rupture of membranes, i.e. women with a complaint of fluid leakage (105 in total) were enrolled. Prior to speculum examination, sagittal and transverse plane views of the fornices and cervix were obtained via transperineal ultrasonography and images were stored. Then the speculum examination for amniotic fluid pooling was performed. Definite diagnoses were made with PAGM-1 assays. After collection of the data, ultrasound images were analyzed by an observer blind to physical examination findings. Hypoechogenic fluid appearance around the cervix and in the fornices was considered positive for preterm premature rupture of membranes (PPROM). Results of physical and ultrasound examination were compared with PAMG-1 test as a reference method. The diagnostic performance of transperineal ultrasound was tested with accuracy parameters and receiver-operating characteristics (ROC) curves. Inter-rater reliability was analyzed with Cohen's kappa.Results: In total, 103 pregnant women were evaluated. The prevalence of PPROM in our study population was 43.14%. At a 5mm diagnostic threshold, the sensitivity and specificity values of transperineal assessment were 95.45% (95% CI: 84.50-99.31%) and 96.55% (95% CI: 88.07-99.48%), respectively, and they were comparable with speculum examination in a population of preterm pregnant women with suspected PPROM (p>0.05). Interobserver reliability analysis with Cohen's kappa has shown good very good agreement with the kappa value of 0.93 (95% CI 0.87-1.00).Conclusions: Transperineal ultrasonography is a novel method that can be used to assess vaginal pooling of amniotic fluid. Ultrasonography offers similar sensitivity and specificity compared with speculum examination and it is non-invasive.