Background: The TDx Fetal Lung Maturity II (FLM II) assay uses amniotic fluid to assess lung maturity of the unborn infant. We investigated common preanalytical factors that influence FLM results, including centrifugation, sample storage, and contamination by whole blood. Methods: We tested 18 specimens after centrifugation and after resuspension by vortex-mixing. We also analyzed 23 specimens stored at -20°C for up to 448 days and then thawed (duplicate measurements), 20 specimens stored at 4°C, and 24 specimens stored at room temperature. In addition, we evaluated the effects of whole blood diluted into 19 different specimens. Results: Centrifugation significantly decreased FLM II results from baseline (P <0.0001), and resuspension returned results to baseline values (P = 0.286). Storage at -20°C produced highly variable results that demonstrated a nonsignificant negative trend associated with storage time. Specimens were stable for 24 h when stored at 4°C and 16 h at room temperature. Blood contamination produced significantly positive differences in results only in specimens with baseline values ≤39 mg/g with a 5.8 mg/g increase in FLM II for every 0.1 × 1012/L increase in the erythrocyte count (slope = 58.4). Conclusions: Resuspension of centrifuged specimens produces clinically valid FLM II results. Results from specimens stored at -20°C can be highly variable and decrease over time. Results from specimens stored at 4°C and at room temperature are stable for 24 and 16 h, respectively. Blood contamination up to 0.03 × 1012 erythrocytes/L is acceptable for FLM II analysis.