Several preclinical studies have shown that prolonged exposure of a young developing brain to anesthetic agents can be detail use of H-scan ultrasound (US) imaging for the detection of early neurotoxic effects associated with isoflurane gas anesthesia in a neonatal animal model. Neonatal mice were exposed to either isoflurane (N=14) or normal oxygen (control, N=4) for 2 h. Animals were then returned to their mothers for 48 h. After euthanasia, neonatal brains were excised and underwent 3D US imaging using a small animal US scanner (Vevo 3100, FUIJIFILM VisualSonics Inc). Raw radiofrequency data was acquired for processing and formation of the 3D H-scan US images. In addition to whole brain segmentation, an atlas-based approach was performed in order to analyze changes in the hippocampus and cortex regions, i.e. behavioral and neurocognitive function-dependent regions of the brain. Experimental ex vivo results indicate that the developing neonatal brain is extremely sensitive to isoflurane exposure. More specifically, mean H-scan US image intensity from the entire brain volume was found to be significantly higher in the isoflurane group when compared to control measurements (11.3pm 0.04 %, p=0.001). The cortex and hippocampal regions of the brain also demonstrated substantial sensitivity towards the neurotoxicity of isoflurane. Compared to control brain tissue measurements, H-scan US image intensity levels were 15.3pm 0.06 % (p=0.01) and 14.9pm 0.06 % (p=0.001) higher in the cortex and hippocampal regions, respectively, after exposure to isoflurane anesthesia for 2 h. Overall, our initial findings reveal that 3D H-scan US imaging can detect differences in neonatal brain tissue exposed to either isoflurane or control gas.