The two main screening tests during pregnancy are those for chromosomal abnormalities and neural tube defects (NTDs). In particular, for NTDs, measurement of maternal serum alpha-fetoprotein (MSAFP) levels early in the second trimester (15-18 weeks of gestation) has been considered the gold standard screening test for the past 4 decades. However, with remarkable technological advancements and the widespread use of ultrasound during those periods, mid-trimester ultrasonography has gradually replaced the role of measuring MSAFP levels as a screening method for NTDs. This change was initiated more about 10 years ago in some countries, which have issued national guidelines to use mid-trimester ultrasonography instead of measuring MSAFP levels as a prenatal screening method for NTDs. However, no significant changes have occurred in Korea, where second-trimester ultrasonography is routinely performed with high-quality equipment. We aimed to provide information regarding the importance of changing the screening method for NTDs from MSAFP measurement to ultrasonography, and to detail methods of implementing mid-trimester ultrasonography for screening purposes. We also share our experience of operating a prenatal diagnostic program for NTDs without using MSAFP for more than 15 years.