Introduction. Serum proteins PAPP-A (pregnancy-associated plasma protein-A) and β-hCG (the β-subunit of human chorionic gonadotropin) levels are components of the 1^st trimester combined screening aimed to detect fetal aneuploidies. In contrast to singleton pregnancies, where the values are well established, this is not the case in twins. Aim: a comparative analysis of serum proteins PAPP-A and β-hCG levels, as well as parameters PAPP-A MoM and β-hCG MoM in the first trimester during pregnancy with dichorionic diamniotic twins that occurred spontaneously or as a result of in vitro fertilization (IVF). Materials and methods. The study group included multiple pregnancies as a result of IVF procedure (n = 100) and spontaneous multiple pregnancies (n = 121) considered as controls. The levels of PAPP-A, β-hCG, PAPP-A MoM and β-hCG MoM were compared using nonparametric Mann–Whitney U test. Results. The PAPP-A MoM level resulted higher in the group of IVF twins (p = 0,007) compared to spontaneous twins: 1.28 [0.97; 1.89] vs. 1.12 [0.87; 1.40] (median [interquartile range]). There were no statistically significant differences in β-hCG levels between the groups of IVF twins and spontaneous twins. Conclusion. The results justify the need for further study of PAPP-A and β-HCG to establish their association with adverse outcomes in both groups of multiple pregnancies.