Amniotic fluid (AF) reflects fetal and placental physiology and provides a valuable window into intrauterine development. However, the extent to which fetal sex influences the mid-gestation AF proteome remains poorly characterized. The present study applied a data-independent acquisition mass spectrometry (DIA-MS) approach to AF samples collected at 16–18 weeks of gestation from pregnancies carrying male and female fetuses. Approximately 1,700 proteins were identified in AF_male and AF_female samples.