Objectives: Our aim in this study was to evaluate the effect of antiepileptic drugs on biochemical components of screening tests. Materials and Methods: This longitudinal case-control study was performed at Ankara City Hospital, Ankara, Turkey, from June 2020 to January 2021. The case group included epileptic singleton pregnant women which were using antiepileptic drugs, between 18 and 44 years of age. The control group included healthy pregnant women between 18 and 44 years old who did not use any drugs. Maternal medical, obstetric, and gynecological history were recorded. The antiepileptic agents that are used in our study were; lamotrigine, levetiracetam, carbamazepine, oxcarbazepine, valproic acid, and clonazepam. For the aneuploidy screening program conducted in our laboratory, the biochemical markers that are analyzed include; free b-hCG and PAPP-A in the first trimester, as well as AFP, HCG, and unconjugated estriol (uE3) in the second trimester. MoM values of these markers were compared between the case and control groups. Results: Fifty-three pregnant women with epilepsy using antiepileptic drugs were compared with 106 healthy pregnant women. The levels of serum-free b-hCG, and PAPP-A were similar between the case and control groups (p = 0.653, p = 0.351). For the second-trimester screening biochemical markers, the maternal serum uE3 and AFP levels expressed as MoM were significantly higher in the epileptic group than in the control patients (p = 0.015 and p = 0.001). Conclusion: Serum marker levels of second-trimester screening tests may be affected by antiepileptic drugs, which may lead to misinterpretation of the risk level. Antiepileptic drug usage should be considered when evaluating screening test results.