Objective: Premature rupture of membranes is defined as the rupture of foetal membranes which may occur at any time before the onset of labour. The purpose of this study was to assess the reliability of cervico-vaginal fluid prolactin level for the diagnosis of premature rupture of membranes and to determine its diagnostic cut off value. Method: A total of 80 pregnant women were included, of which, 40 pregnant women between 20 to 41 weeks of gestation, complaining of leaking PV with amniotic fluid pooling +, nitrazine test +, and fern test +, were included in the study group and 40 pregnant women with no complaints were included in control group. All patients underwent USG for AFI calculation and vaginal washing fluid sampling for prolactin assay. Results: For PROM 34.35 µIU/ml (area under the curve (AUC) = 0.953) was the optimal cut-off value of vaginal prolactin, with a sensitivity of 92.5% and a specificity of 87.5%. It had PPV of 88.09%, NPV of 92.11% and accuracy of 90%. TheStandard Errorwas0.024. Conclusion: Vaginal washing fluid prolactin is a suitable marker for the diagnosis of PROM which can be used as an adjunctive test particularly in ambiguous cases of PROM.