Clinical and Laboratory Standards Institute document I/LA25-A2--Maternal Serum Screening; Approved Standard--Second Edition is written for clinical laboratorians who participate in prenatal screening for open neural tube defects and trisomy 21 (Down syndrome) involving alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), unconjugated estriol (uE3), inhibin A, and/or pregnancy-associated plasma protein-A (PAPP-A) measurements, as well as for clinicians and manufacturers who have a direct interest in the tests. First-trimester screening (including nuchal and ultrasound measurements) and integrated first- and second-trimester screening are emphasized. The standard is intended to present necessary considerations: preanalytical, analytical, and postanalytical (preexamination, examination, and postexamination); and to ensure the reliability of the tests, including the risk calculation, the outcome evaluation, and the accuracy of the information management. If properly applied, the five biochemical determinations and the risk calculations can contribute constructively to the field of prenatal screening and to the welfare of pregnant women and the fetus.