During a maternal serum screening, the mother's blood is checked for a combination of different markers: alpha-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG) make up the standard tests, known together as the "triple test." Sometimes a marker called inhibin A is added, making the "quadruple screen." These tests are independent measurements, and when taken along with the maternal age (discussed below), can calculate the risk of having a baby with Down syndrome. Over the last fifteen years, these were done in the 15th to 18th week of pregnancy. Recently, another marker called PAPP-A was found to be of use even earlier.

• Alpha-fetoprotein (AFP) is made in the part of the womb called the yolk sac and in the fetal liver, and some amount of AFP gets into the mother's blood. In neural tube defects, the skin of the fetus is not intact and so larger amounts of AFP is measured in the mother's blood. In Down syndrome, the AFP is decreased in the mother's blood, presumably because the yolk sac and fetus are smaller than usual.
• Estriol is a hormone produced by the placenta, using ingredients made by the fetal liver and adrenal gland. Estriol is decreased in the Down syndrome pregnancy. This test may not be included in all screens, depending on the laboratory.
• Human chorionic gonadotropin hormone (HCG) is produced by the placenta and is used to test for the presence of pregnancy. A specific smaller part of the hormone, called the beta subunit, is increased in Down syndrome pregnancies.
• Inhibin A is a protein secreted by the ovary and is designed to inhibit the production of the hormone FSH by the pituitary gland. The level of inhibin A is increased in the blood of mothers of fetuses with Down syndrome.
• PAPP-A, which stands for pregnancy-associated plasma protein A, is produced by the covering of the newly fertilized egg. In the first trimester, low levels of this protein are seen in Down syndrome pregnancies.

A very important consideration in the screening test is the age of the fetus (gestational age). The correct analysis of the different components depends on knowing the gestational age precisely. The best way to determine that is by ultrasound.

Once the blood test results are determined, a risk factor is calculated based on the "normal" blood tests for the testing laboratory. The average of normals is called the "population median." Test results are sometimes reported to doctors as "Multiples of the Median (MoM)." The "average" value is therefore called 1.0 MoM. Down syndrome pregnancies have lower levels of AFP and estriol, so their levels would be below the average, and therefore less than 1.0 MOM. Likewise, hCG in a Down syndrome pregnancy would be greater than 1.0 MoM. In the serum screening, the lab reports all results in either this way or as a total risk factor calculated by a software program.