Myelomeningocele (MMC), the most severe form of spina bifida (SB), causes neurological deficit. Injury to the spinal cord is thought to begin in utero. We investigated whether brain-specific proteins (BSPs) would enable us to monitor the development of MMC-related tissue damage during pregnancy in an animal model with naturally occurring SB (curly tail/loop tail mouse; n = 256). Amniotic fluid levels of neurofilament heavy chain (NfH), glial acidic fibrillary protein (GFAP) and S100B were measured by standard ELISA techniques. The amniotic fluid levels of all BSPs were similar in SB and control mice on embryonic day (E) 12.5 and 14.5, whereas a significant increase was observed for GFAP in SB mice on E16.5. Levels of all BSPs were significantly increased in SB mice on E18.5. The rapid increase in GFAP, paralleled by a moderate increase in NfH and S100B, suggests that spinal cord damage starts to accelerate around E16.5. The macroscopic size of the MMC was related to NfH level on E16.5 and E18.5, suggesting that axonal degeneration is most severe in large MMC. Amniotic fluid BSP measurements may provide important information for balancing the risks and benefits to mother and child of in utero surgery for MMC.