Abstract Our recent exploratory proteomic study suggested increased levels of neutrophil-gelatinase associated lipocalin (P80188, NGAL_HUMAN) due to microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) in women with preterm prelabour rupture of membranes. In this study, we verified the proteomics findings by assessing the amniotic fluid NGAL by ELISA in the original exploratory cohort. The NGAL level was significantly higher in women positive for both MIAC and HCA compared to women with both conditions ruled out (median 75.1 ng/ml vs. 27.9 ng/ml; p<0.0001). For independent validation and to assess NGALs potential to stratify women positive for both MIAC and HCA from women in whom at least one of these conditions was absent, we subsequently designed a retrospective replication cohort. Significantly higher NGAL levels were found in women positive for both MIAC and HCA (median 65.9 ng/ml vs. 34.2 ng/ml; p=0.0061). Significantly higher levels of NGAL were confirmed only in strata below 32 weeks of gestation. Based on the observed likelihood ratio, the best predictive cutoff level (47.1 ng/ml) was evaluated in both cohorts. Data from the verification cohort implied that NGAL is a valuable clinical marker for revealing MIAC leading to HCA; however, this potential was not replicated in the replication cohort.