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Abstract Background Noninvasive prenatal testing (NIPT) is the testing of blood samples from pregnant women to screen for fetal risk of chromosomal disorders. Even though in vitro hemolysis of blood specimens is common in clinical laboratories, its influence on NIPT has not been well investigated. Methods Peripheral blood samples were collected from 205 pregnant women and categorized according to the concentration of free hemoglobin in the plasma. After performing NIPT using massively parallel sequencing, the quality control metrics were analyzed and compared with samples that did not undergo hemolysis or samples redrawn from the same women. Results The specimens were divided into four groups based on the concentration of free hemoglobin: Group I (0–1 g/L, n = 53), Group II (1–2 g/L, n = 97), Group III (2–4 g/L, n = 30), and Group IV (> 4 g/L, n = 25). There was no significant difference in the quality control metrics of clinical samples with slight or moderate hemolysis (Group II and III). However, samples with severe hemolysis (Group IV) showed a significantly increased rate of duplicated reads (duplication rate) and fetal fraction, as well as decreased library concentration compared with samples without hemolysis. Moreover, the increase in fetal fraction caused by hemolysis was confirmed by redrawing blood samples in Group IV. Conclusion For NIPT using massively parallel sequencing, samples with slight or moderate hemolysis (≤ 4 g/L) are acceptable. However, careful consideration should be taken regarding the use of severely hemolyzed samples (> 4 g/L), since they might increase the risk of test failure.