Preeclampsia is a serious disorder of human pregnancy and its pathogenic origin is defective placental development during early pregnancy. However, owing to the lack of reliable early detection methods, preeclampsia is not diagnosed until later in pregnancy and premature delivery remains the sole effective therapy. We cloned HtrA3, a serine protease with high expression during placental development in the mouse, rhesus monkey and human. HtrA3 exists in two isoforms [long (HtrA3-L) and short (HtrA3-S)] and both are expressed in human placenta. The two HtrA3 isoforms are identical except that the HtrA3-L has an extra C-terminal domain (the PDZ domain). We further established that in women, placental HtrA3 was secreted into the maternal circulation with its serum profile reflecting placental production. We also reported that maternal serum HtrA3 levels at the end of first trimester significantly differed between women who subsequently proceeded with normal pregnancy or developed preeclampsia. In this study, using highly specific HtrA3 monoclonal antibodies that we generated in our laboratory, we established and fully validated ELISAs suitable for the detection of total HtrA3 and HtrA3-L in human serum. We then conducted a retrospective study to determine serum HtrA3 at 11-13 weeks of gestation in a cohort of singleton pregnancies that were subsequently proceeded with normal pregnancy (control n=292), or complicated with preeclampsia (n= 41) or small for gestational age (SGA, n = 71). Compared to controls, SGA pregnancies showed significantly lower levels of total HtrA3, with no difference in HtrA3-L. In contrast, the preeclamptic cases compared to controls had significantly higher levels of HtrA3-L, with no difference in total HtrA3. In addition, a sub-set of preeclampsia, early-onset preeclampsia, showed an altered ratio of HtrA3-L over total HtrA3. These data support the potential utility of serum HtrA3 for early detection of preeclampsia and SGA.