There is a lack of standardized measurement tools globally to assess knowledge, attitudes, and perceptions of expecting women toward prenatal screening. The purpose of this systematic review was to identify reasons women pursue or decline prenatal screening and compare the strengths and limitations of available measurement tools used to assess pregnant women’s perceptions, knowledge, and attitudes toward prenatal screening. This review followed the five-step York methodology by Arksey and O’Malley and incorporated recommendations from the Preferred Reporting Items for Systematic Reviews and Meta-Analysis checklist for the extraction, analysis, and presentation of results. The five steps consisted of: (1) identification of the research questions; (2) searching for relevant studies; (3) selection of studies relevant to the research questions; (4) data charting; and (5) collation, summarization, and reporting of results. Four online databases (PubMed, Embase, Web of Science, and Cochrane Library) were selected after the librarian’s development of a detailed search strategy. The Rayyan platform was used between June 2023 and August 2023 to epitomize the articles produced from our search. A total of 68 eligible studies were included in the analysis. The top five major reasons for declining prenatal screening uptake included (1) being unsure of the risk of prenatal screening and harm to the baby or miscarriage ( n = 15), (2) not considering action such as termination of pregnancy for prenatal screening to be considered as necessary ( n = 14), (3) high cost ( n = 12), (4) lack of knowledge about testing procedures and being anxious about the test ( n = 10), and (5) being worried about probability of false negative or false positive results ( n = 6). Only 32 studies utilized scientifically validated instruments. Difficulties in capturing representative, adequately sized samples inclusive of diverse ethnicities and demographics were pervasive. Findings highlight the need for rigorous validation of research measurement methodologies to ensure the accuracy and applicability of resulting data regarding the assessment of prenatal screening perceptions, knowledge, and attitudes across diverse female populations. Registration: N/A.