Down Syndrome or Trisomy 21 (T21) is a complex genetic disease characterized by the presence of an extra chromosome 21, which leads to multiple clinical features and manifestations that severely affect the patient's quality of life. Various methods of prenatal screening have been developed over time, allowing informed decision-making. However, a common drawback of the current methods for detecting T21 is their invasive nature. Over the past years, mass-spectrometry-based omics technologies have become a key tool for discovering biomarkers for the prenatal screening of T21, particularly focusing on proteins, peptide sequences, or metabolites in samples, like amniotic fluid, umbilical cord blood, and others. Recently, there has been a noticeable shift towards using less invasive biological sample types (e.g., maternal serum, plasma, and urine) reflecting a growing interest in non-invasive methods for prenatal screening. These advances aim to improve the sensitivity and accuracy for T21 detection while reducing the risks associated with more invasive procedures. The first section of this paper offers an in-depth review of studies utilizing mass-spectrometry-based omics for the prenatal screening of T21. This part provides an overview of the methodologies employed and their key findings. Instead, the subsequent section offers a comprehensive examination of the differentially expressed proteins (DEPs) and metabolites (DEMs) reported in the literature in T21 prenatal screening. Additionally, pathway analysis is carried out to explore the biological pathways that these molecules are involved in and how they relate to the clinical features of the syndrome. These findings aim to guide future research in the field and foster the development of more advanced, less invasive prenatal screening techniques for T21.