Aim: To estimate the optimal level of vaginal fluid creatinine to detect rupture of membranes (ROM). Materials and methods: A total of 300 pregnant women were recruited for the study. Group I consisted of 100 women with a diagnosis of ruptured amniotic membranes confirmed by sterile speculum examination. Group II consisted of 100 women in whom a diagnosis of ROM was suspected but not confirmed by speculum examination. Group III consisted of 100 pregnant women without any complaints taken as a control group. In all 300 women, vaginal fluid washing for creatinine sample was taken. The parameters (age, parity, gestational age at the time of sample collection, amniotic fluid index (AFI), and vaginal fluid creatinine) were compared with one-way analysis of variance (ANOVA) and ScheVe multiple comparison test. Receiver operating characteristic (ROC) curve analysis was used to establish an optimal cut-off concentration. Results: The optimal cut-off value of vaginal fluid creatinine to detect ROM arrived based on ROC curve analysis was ≥0.3 mg/dL. The mean vaginal fluid creatinine in group I was 1.097 mg/dL, in group II was 0.3 mg/dL, in group III was 0.068 mg/dL. This study demonstrates that vaginal fluid creatinine could accurately diagnose a ROM with a sensitivity, specificity, positive predictive value, and negative predictive value of 98.36, 100, 100, and 97.14%, respectively.