Prenatal diagnosis aims either to provide the reassurance to the couples at risk of having an affected child by timely appropriate therapy or to give the parents a chance to decide the fate of the unborn babies with health problems. Invasive prenatal diagnosis (IPD) is accurate, however, carrying a risk of miscarriage. Non-invasive prenatal diagnosis (NIPD) has been developed based on the existing of fetal genetic materials in maternal circulation; however, a minority fetal DNA in majority maternal background DNA hinders the detections of fetal traits. Different protocols and assays, such as homogenous MassEXTEND (hME), single allele base extension reaction (SABER), precise measuring copy number variation of each allele, and quantitative methylation and expression analysis using the high-throughput sensitive matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), allow NIPD for single gene disorders, fetal blood group genotyping and fetal aneuploidies as well as the development of fetal gender-independent biomarkers in maternal circulation for management of pathological pregnancies. In this review, we summarise the use of MALDI-TOF MS in prenatal genomics.