The protocol described in the present article aims to validate the GLU test, a test of mid-pregnancy vaginal microbiome, for PTB risk prediction in pregnant First Nations women. Preterm birth (PTB; birth before 37 completed weeks gestation) is associated with a higher risk of adverse neonatal outcomes. First Nations communities are affected by increasing PTB rates, highest in remote communities, reaching 23%. Being able to predict women at high risk of PTB is one of the greatest challenges of our time. No reliable clinical predictors of PTB risk currently exist, beyond a previous history. Spontaneous PTB (sPTB) is highly associated with microbial infection. Recently, a Western Australian research team developed an innovative mid-pregnancy vaginal microbial DNA test, the ‘Gardnerella, Lactobacillus, Ureaplasma’ (GLU) test, capable of predicting up to 45% of sPTB cases. However, this test has only been validated in predominantly Caucasian pregnant women. The protocol described aims to validate the GLU test in pregnant First Nations women and where applicable, make modifications to this test to improve sensitivity and specificity within this population.