Background and Purpose To identify differentially expressed peptides in cerebrospinal fluid (CSF) of neonates with hypoxic-ischemic brain injury (HIBD) or controls, which may give a foundation for finding new promising drugs of neonatal HIBD. Experimental Approach CSF samples were collected from neonates with HIBD (n=4) or controls (n=4). ITRAQ LC-MS/MS was used to identify differentially expressed peptides between two groups. Effects of the peptide from heat shock protein 90-alpha (HSP90α/HSP90AA1) on cell viability and pyroptosis under oxygen and glucose deprivation (OGD) were analyzed using cell counting kit-8 (CCK-8) assay and Annexin V-fluorescein isothiocyanate (FITC) Assay. Expressions of NLRP3, ASC and Caspase-1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Key Results Compared with the control group, one peptide significantly up-regulated and thirty-four peptides significantly down-regulated in the CSF of neonates with HIBD. A fragment of HSP90α/HSP90AA1 is the 2671.5Da peptide (HSQFIGYPITLFVEKER), one of the down-regulated peptides in neonatal HIBD. This peptide, we named it HIBDAP, inhibited pyroptosis of PC12 cells under OGD by suppressing expressions of NLRP3, ASC and Caspase-1. Conclusion and Implications The results of our study identified the characterization and expression profiles of peptides in CSF of neonatal HIBD. Several meaningful peptides such as HIBDAP may play significant roles in neonatal HIBD and may provide new therapeutic targets for neonatal HIBD.