Introduction: Altered placental expression of high temperature requirement factor A1 (HtrA1) is implicated in abnormal trophoblastic invasion and endothelial dysfunction in pre-eclampsia (PE). Serum levels of HtrA1 have been proposed as a novel biomarker to improve the prediction of PE. This study assesses serum HtrA1 levels in prospectively collected samples of women who developed PE compared to normotensive pregnancies. Methods: This was a case-control study of serum HtrA1 levels in second and third trimester samples in women who later developed preterm or term PE compared to controls. Overall, 300 serum samples were drawn from a prospective observational study of adverse pregnancy outcomes in three different gestational age windows (19–24, 30-34 and 35–37 weeks) at the Fetal Medicine Research Institute, King's College Hospital, London. Serum HtrA1 levels were determined by enzyme-linked immunosorbent assay (ELISA) by a blinded laboratory professional. Median HtrA1 MoM values, adjusted for gestational age and maternal characteristics, were compared between cases and controls at each gestational age group. Results: Women who later developed PE, compared to controls, had significantly higher maternal weight and more frequently had chronic hypertension or a history of PE in a previous pregnancy. In normotensive pregnancies, serum HtrA1 increased with increasing gestational age, whereas, in PE pregnancies HtrA1 levels remained stable, but were not significantly different from control pregnancies at any gestational age. Discussion: Serum HtrA1 levels are not significantly different in women who develop PE compared to controls.