Using mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to explore the pathophysiology biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented 4-plex N, N-dimethyl leucine (DiLeu) isobaric labeling reagents in a longitudinal study to quantify CSF proteins from children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. Results from this study identified differentially expressed proteins in CSF samples collected from week 5, week 10-14 and week 24-28 compared with samples before Chemo treatment. 84 proteins were significantly altered while Gene Ontology analysis revealed their involvement in various biological processes associated with neurologic disorders. Results from this study will be helpful for monitoring central nervous system (CNS)-specific proteins and developing preventive strategies for CNS injury in response to Chemo.