Objective: To determine if quantitative fetal fibronectin (qFFN) in addition to transvaginal ultrasound (TVU) cervical length (CL) measurement between 180/7 and 236/7 weeks would be predictive of spontaneous preterm birth (sPTB) at <350/7 weeks among asymptomatic high-risk women as defined by those with prior sPTB > 200/7 weeks.Material and methods: A prospective observational study of asymptomatic women with singleton gestations between 180/7 and 236/7weeks and one or more prior SPTB was performed. Women at their anatomy scan who opted into universal CL screening were enrolled. At enrollment, a vaginal speculum exam was performed to collect cervico-vaginal fluid from the posterior fornix using fetal fibronectin (FFN) swab. These women were then followed until delivery. Women with multiple gestations, rupture of membranes, vaginal bleeding, intercourse, or vaginal exam within 48 h of enrollment were excluded. Physicians were blinded to the qFFN levels, but the CL measurements were made available. The primary outcome was sPTB < 350/7 weeks.Results: Of the 105 asymptomatic women with prior sPTB who were prospectively enrolled, 19 (18.1%) had recurrent sPTB < 370/7 weeks. None of the sPTB were iatrogenic. Using receiver-operating characteristic curves, qFFN ≥ 10 ng/mL had the highest sensitivity with subsequent lowest false negative rate, while FFN ≥ 50 ng/mL was identified as being the best balance of sensitivity and false positive rate for predicting sPTB < 350/7 weeks. As compared with CL ≤ 25 mm alone, with the use of CL ≤ 25 mm or qFFN ≥ 50 ng/mL as screening criteria for prediction of SPTB < 350/7 weeks, sensitivity improved from 18.2 to 63.6%, specificity decreased from 96.8 to 82.1%, positive predictive value (PPV) decreased from 40.0 to 29.2%, negative predictive value (NPV) marginally improved from 91.1 to 95.1%.Conclusion: In women with singleton gestations with prior SPTB, qFFN can be used as an adjunct to triage patients who are found to have a shortened cervix. Sensitivity and NPVs improved with the addition of qFFN to TVU CL screening alone in women with singleton gestations with prior SPTB. However, specificity and PPVs decreased.